Fumarylacetoacetate hydrolase (fah)-deficient and immunodeficient rats and uses thereof

ABSTRACT

Described herein are rats with a hepatic deficiency comprising decreased function, activity, or expression of an enzyme in the tyrosine catabolic pathway (such as fumarylacetoacetate hydrolase), and methods of using the same for in vivo engraftment and expansion of heterologous hepatocytes, such as human hepatocytes, analysis of human liver disease, and analysis of xenobiotics. Also disclosed is the use of immunodeficient rats for the engraftment and expansion of heterologous hepatocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 14/241,316,which is the U.S. National Stage of PCT/US2012/052306, filed Aug. 24,2012, which claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 61/527,865, filed Aug. 26, 2011,FUMARYLACETOACETATE HYDROLASE (FAH)-DEFICIENT ANIMALS AND USE THEREOF,each of which is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under grantnumber 1DP2OD008396-01, awarded by the National Institutes of Health.The U.S. government has certain rights in the invention.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequenceare presented in accordance with 37 C.F.R. 1.822. Only one strand ofeach nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. TheSequence Listing is submitted as an ASCII text file, created on Aug. 24,2012, 22 KB, which is incorporated by reference herein. In theaccompanying sequence listing: SEQ ID NO:1 is the sequence of a portionof the rat FAH protein; SEQ ID NO:2 is the nucleotide sequence of therat Fah gene targeting cassette; SEQ ID NOs:3-20 are nucleotidesequences of primers; SEQ ID NO:21 is the nucleotide sequence of aportion of the wild type Fah rat gene; SEQ ID NO:22 is the amino acidsequence of the wild type FAH protein; SEQ ID NOs:23-25 are amino acidsequences of mutants m1-m3; SEQ ID NO:26 is the amino acid sequence of aportion of the wild type Il2rg gene; SEQ ID NO:27 is the amino acidsequence of a portion of the wild type Rag2 gene; SEQ ID NO: 28 in theamino acid sequence of SEQ ID NO:1; and SEQ ID NO:29 is the amino acidsequence of anakinra.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure concerns rat models having a hepatic deficiencyand/or an immunodeficiency and their use. This disclosure also relatesto methods of using such animal models, including expanding heterologoushepatocytes, such as human hepatocytes, in such animal models.

2. Description of Related Art

The liver is the principal site for the metabolism of xenobioticcompounds including medical drugs. Because many hepatic enzymes arespecies-specific, it is necessary to evaluate the metabolism ofcandidate pharmaceuticals using cultured primary human hepatocytes ortheir microsomal fraction. While microsomal hepatocyte fractions can beused to elucidate some metabolic functions, other tests depend on livinghepatocytes. Some compounds, for example, induce hepatic enzymes andthus their metabolism changes with time. To analyze enzyme induction,hepatocytes must be not only viable, but fully differentiated andfunctional.

Human hepatocytes are widely used by the pharmaceutical industry duringpreclinical drug development. Their use is mandated by the FDA as partof drug development. For drug metabolism and other studies, hepatocytesare typically isolated from cadaveric organ donors and shipped to thelocation where testing will be performed. The condition (viability andstate of differentiation) of hepatocytes from cadaveric sources ishighly variable and many cell preparations are of marginal quality. Theavailability of high quality human hepatocytes is further hampered bythe fact that they cannot be significantly expanded in tissue culture.After plating, the cells survive but do not divide. Hepatocytes fromreadily available mammalian species, such as the mouse, are not suitablefor drug testing because they have a different complement of metabolicenzymes and respond differently in induction studies. Immortal humanliver cells (hepatomas) or fetal hepatoblasts are also not an adequatereplacement for fully differentiated adult cells. Human hepatocytes arealso necessary for studies in the field of microbiology. Many humanviruses, such as viruses that cause hepatitis, cannot replicate in anyother cell type.

Moreover, bioartificial liver assist devices, which use hepatocytes exvivo, have been used to support patients in acute liver failure. Inaddition, several clinical trials of hepatocyte transplantation havebeen carried out, which provided proof-of-principle that hepatocytetransplantation can be beneficial. Currently, human hepatocytes cannotbe expanded significantly in culture. Hepatocytes derived from stemcells in culture are immature and generally lack full functionality.Therefore, all hepatocytes in use today are derived from human donors,either cadaveric or surgical specimens, which significantly limitshepatocyte availability. If enough human hepatocytes were available,bioartificial liver assist devices would become a viable technology andhuman hepatocyte transplantation could find wide-spread use. Given theselimitations, methods of expanding primary human hepatocytes are highlydesirable. There is also a need in the animal health industry forprocesses to expand hepatocytes from other species, such as dogs,horses, etc. for research and study.

SUMMARY

Described herein are rats that have utility for a variety of purposes,including for the expansion of hepatocytes from other species(particularly humans), and as animal models of liver diseases, includingcirrhosis, hepatocellular carcinoma and hepatic infection. In general,the rats have dysfunctional livers (“hepatic deficiency”), induced liverdamage, and/or an immunodeficiency, as described in more detail herein.

Provided herein are methods of expanding heterologous hepatocytes, andparticularly human hepatocytes in vivo. In some embodiments, the methodincludes transplanting heterologous hepatocytes (or hepatocyteprogenitors) into an Fah-deficient rat and allowing the heterologoushepatocytes to expand. In some cases, the Fah-deficient rat isimmunosuppressed or immunodeficient. In some embodiments, theFah-deficient rat is administered a vector encoding urokinase prior totransplantation of the heterologous hepatocytes.

Also provided are methods of expanding heterologous hepatocytes in vivoby transplanting heterologous hepatocytes (or hepatocyte progenitors)into an immunodeficient rat and allowing the heterologous hepatocytes toexpand. In some embodiments, the heterologous hepatocytes are allowed toexpand for at least about 2, 4, 5, 6, 7, 8, weeks, or several months.

Isolated hepatocytes (such as human hepatocytes) expanded in andcollected from Fah-deficient and/or immunodeficient rats are alsoprovided by the present disclosure.

Also provided are genetically modified rats whose genomes are homozygousfor a disruption in the Fah gene such that the disruption results inloss of expression of functional FAH protein, wherein the rats exhibitdecreased liver function. In some examples, the Fah-deficient ratsfurther include transplanted heterologous hepatocytes. In some cases,the Fah-deficient rat is immunosuppressed.

Further provided are isolated rat embryonic stem cells comprising adisruption in the Fah gene.

Also provided are methods of producing an Fah-deficient rat cell byvarious methods including homologous recombination using the AAV-DJvector, as well as TALENs.

Methods of using the rat models for in vivo analysis of human liverdisease, drug candidates, and xenobiotics are also provided.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of the wild type rFAH gene including intron 2-3and intron 3-4, along with the nucleotide sequence (SEQ ID NO:1) withcorresponding amino acid sequence (SEQ ID NO:28) and PCR primers. Thesequences adjacent the introns in the schematic indicate the sequenceincluded in the rFah KO targeting cassette. The outside sequencesindicate sequences that lie outside of the target sequence, but are partof the rFah gene.

FIG. 2 is a schematic diagram showing the rat Fah targeting constructcontaining the 1.5 kb 5′ homology region and the 1.5 kb 3′ homologyregion flanked by the 1.7 kb selection cassette PGK:NEO;

FIG. 3 is a schematic diagram of the rat Fah gene with the integratedtargeting KO cassette containing the PGK:NEO sequence;

FIG. 4 is a schematic of the rFah gene with the integrated targeted KOcassette containing the PGK:NEO sequence. The arrows indicate the primerbinding sites and the brackets indicate the entire PCR product. The 5′and 3′ PCR primer pairs contain one primer that annealed to the rFahgene outside of the targeted sequence and a second primer that annealedto the PGK:NEO sequence. Each PCR reaction contained 0.1 ng of gDNA and2 μM of each PCR primer. Denaturing, annealing and extensiontemperatures along with cycle numbers were optimized for each primerset;

FIG. 5 shows (A) a photograph of the 5′ and 3′ PCR reactions for eachpotential rFah KO ES clone 11-19. The predicted molecular weights ofeach primer pair are indicated. Clones 14, 17 and 19 do not appear tocontain the integrated rFah KO cassette; and (B) a photograph of PCRproducts from all of the ES clones using rFah primers outside of theintegration site;

FIG. 6 shows (A) a schematic of the rFah gene WT and KO genome of thetargeted region. The arrows and bracket indicate the ³²P-labeled PCRproduct used for hybridizing the southern blot; and (B) a southern blotimage. Fifteen μg of genomic DNA from each ES clones was digested withBgl II restriction endonuclease overnight at 37° C. The digestedproducts were electrophoresed on a 0.7% Agarose:TBE gel, the DNA wasexposed to acid depurination prior to capillary transfer to nylonmembrane with alkaline transfer buffer. The membrane was hybridized with³²P labeled probe from the indicated PCR product in A and B;

FIG. 7 shows images of (A) FAH positive staining (red) of a cluster ofhuman hepatocytes inside the liver of rat 29; and (B) FAH positivestaining (red) of human hepatocytes inside the liver of FRG KO mousecontrol;

FIG. 8 is (A) a schematic of a pair of TALENs designed to target anddisrupt sequences in exon 3 of the rat Fah gene (GenBank AccessionNM_017181); (B) the wild-type TALEN target sequence with specific TALENbinding sites underlined and in capital letters (SEQ ID NO:21);

FIG. 9 is the amino acid sequence of the wild type and mutationsidentified in Example 6 SEQ ID NOs:22-25);

FIG. 10 is the TALENs targeting coding sequences of the rat Rag2 and thechromosome X-linked Il2rg genes injected into SS and SD embryos todisrupt both genes in both strains in Example 6 (SEQ ID NO:26). The wildtype sequence of each target site is shown with the TALEN monomerbinding sites underlined;

FIGS. 11 and 12 show immune phenotyping of null animals(SS-Il2rg-m1^(−/y) and SS-Rag2-m1^(−/−) males) revealing deficiencies ofCD3+ T-cells (green circles), CD45RA+ B-cells (blue circles) in bothstrains, and CD3−/CD161a^(HIGH) NK-cells (red circles) specifically inthe Il2rg null animals;

FIG. 13 is a table of genotypes of live born rat pups from Fahheterozygous breedings;

FIG. 14 is Trichrome staining of fixed tissue from a 9-week oldSS-Fah-m1^(−/−) after 8 days of NTBC withdrawal showing evidence ofearly stages of liver dysfunction evident as (arrows) compared to acontrol SS inbred animal of a similar age on NTBC; and

FIG. 15 is Trichrome staining of fixed tissue from a 9-week oldSS-Fah-m1^(−/−) after 8 days of NTBC withdrawal showing evidence ofearly stages of renal injury compared to a control SS inbred animal of asimilar age on NTBC.

DETAILED DESCRIPTION I. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitatereview of the various embodiments of the disclosure, the followingexplanations of specific terms are provided:

AAV-DJ vector: An adeno-associated virus (AAV) packaging helper thatexpresses a hybrid capsid containing AAV2, AAV5 and AAV8 capsid proteinsand the AAV2 rep protein. (Grimm et al., J Virol 82:5887, 2008; U.S.Pat. No. 7,588,772; U.S. Patent Application Publication No.2010/0047174).

Administration: To provide or give a subject an agent, such as atherapeutic agent, by any effective route. Exemplary routes ofadministration include, but are not limited to, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), oral, intraductal, sublingual, rectal, transdermal,intranasal, vaginal and inhalation routes.

Agent that inhibits or prevents or avoids the development of liverdisease: A compound or composition that when administered to anFah-deficient rat, prevents, avoids, delays or inhibits the developmentof liver disease in the animal. Liver disease or liver dysfunction ischaracterized by any one of a number of signs or symptoms, including,but not limited to an alteration in liver histology (such as necrosis,inflammation, fibrosis, dysplasia or hepatic cancer), an alteration inlevels of liver-specific enzymes and other proteins (such as aspartateaminotransferase, alanine aminotransferase, bilirubin, alkalinephosphatase and albumin), plasma or urinary succinylacetone (SA), orgeneralized liver failure. In some embodiments, the agent that inhibitsliver disease is a pharmacologic inhibitor of 4-OH-phenylpyruvatedioxygenase, such as 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1,3cyclohexanedione (NTBC) or methyl-NTBC. In one non-limiting example, theagent that inhibits liver disease is NTBC.

Anakinra: An interleukin-1 (IL-1) receptor antagonist. Anakinra blocksthe biologic activity of naturally occurring IL-1 by competitivelyinhibiting the binding of IL-1 to the IL-1 receptor, which is expressedin many tissues and organs. IL-1 is produced in response to inflammatorystimuli and mediates various physiologic responses, includinginflammatory and immunologic reactions. Anakinra is a recombinant,non-glycosylated version of human IL-1RA (IL-1 receptor antagonist)prepared from cultures of genetically modified Escherichia coli. Theanakinra protein is 153 amino acids and has a molecular weight ofapproximately 17.3 kD and differs from native human IL-1RA in that ithas a single methionine residue on its amino terminus (the amino acidsequence of anakinra is set forth herein as SEQ ID NO:29). Anakinra isalso known as KINERET™.

Azathioprine: An immunosuppressant that is a purine synthesis inhibitor,inhibiting the proliferation of cells, especially leukocytes. Thisimmunosuppressant is often used in the treatment of autoimmune diseasesor organ transplant rejection. It is a pro-drug, converted in the bodyto the active metabolites 6-mercaptopurine (6-MP) and 6-thioinosinicacid. Azathioprine is produced by a number of generic manufacturers andas branded names (Azasan™ by Salix; Imuran™ by GlaxoSmithKline; Azamun™;and Imurel™).

Biological sample: A sample obtained from cells, tissue or bodily fluidof a subject, such as peripheral blood, serum, plasma, cerebrospinalfluid, bone marrow, urine, saliva, tissue biopsy, surgical specimen, andautopsy material. Also referred to herein as a “sample.”

Cirrhosis: Refers to a group of chronic liver diseases characterized byloss of the normal microscopic lobular architecture and regenerativereplacement of necrotic parenchymal tissue with fibrous bands ofconnective tissue that eventually constrict and partition the organ intoirregular nodules. Cirrhosis has a lengthy latent period, usuallyfollowed by sudden abdominal pain and swelling with hematemesis,dependent edema, or jaundice. In advanced stages there may be ascites,pronounced jaundice, portal hypertension, varicose veins and centralnervous system disorders that may end in hepatic coma.

Collecting: As used herein, “collecting” expanded heterologoushepatocytes refers to the process of removing the expanded hepatocytesfrom a rat that has been injected or transplanted with isolatedheterologous hepatocytes (also referred to as a recipient rat).Collecting optionally includes separating the hepatocytes from othercell types. In one embodiment, the expanded heterologous hepatocytes arecollected from the liver of a rat.

Common-γ chain of the interleukin receptor (Il2rg): A gene encoding thecommon gamma chain of interleukin receptors. Il2rg is a component of thereceptors for a number of interleukins, including IL-2, IL-4, IL-7 andIL-15 (Di Santo et al. Proc. Natl. Acad. Sci. U.S.A. 92:377-381, 1995).Animals deficient in Il2rg exhibit a reduction in B cells and T cellsand lack natural killer cells. Also known as interleukin-2 receptorgamma chain.

Cryopreserved: As used herein, “cryopreserved” refers to a cell (such asa hepatocyte) or tissue that has been preserved or maintained by coolingto low sub-zero temperatures, such as 77 K or −196° C. (the boilingpoint of liquid nitrogen). At these low temperatures, any biologicalactivity, including the biochemical reactions that would lead to celldeath, is effectively stopped.

Cyclosporin A: An immunosuppressant compound that is a non-ribosomalcyclic peptide of 11 amino acids produced by the soil fungus Beauverianivea. Cyclosporin A is used for the prophylaxis of graft rejection inorgan and tissue transplantation. Cyclosporin A is also known ascyclosporine and ciclosporin.

Decreased liver function: An abnormal change in any one of a number ofparameters that measure the health or function of the liver. Decreasedliver function is also referred to herein as “liver dysfunction.” Liverfunction can be evaluated by any one of a number of means well known inthe art, such as, but not limited to, examination of liver histology andmeasurement of liver enzymes or other proteins. For example, liverdysfunction can be indicated by necrosis, inflammation, fibrosis,oxidative damage or dysplasia of the liver. In some instances, liverdysfunction is indicated by hepatic cancer, such as hepatocellularcarcinoma. Examples of liver enzymes and proteins that can be tested toevaluate liver dysfunction include, but are not limited to, alanineaminotransferase (ALT), aspartate aminotransferase (AST), bilirubin,alkaline phosphatase and albumin. Liver dysfunction also can result ingeneralized liver failure. Procedures for testing liver function arewell known in the art, such as those taught by Grompe et al. (Genes Dev.7:2298-2307, 1993) and Manning et al. (Proc. Natl. Acad. Sci. U.S.A.96:11928-11933, 1999).

Deficient: As used herein, “Fah-deficient” or “deficient in Fah” refersto an animal, such as a rat, having a substantial decrease in, or theabsence of, FAH enzyme production or activity, for example an animalhaving a disruption in the Fah gene (such as an insertion, deletion orone or more point mutations), which results in a substantial decreasein, or the absence of, Fah mRNA expression and/or functional FAH enzymeactivity. As used herein, the term “loss of expression” of functionalFAH protein does not refer to only a complete loss of expression, butalso includes a substantial decrease in expression of functional FAHprotein, such as a decrease of about 80%, about 90%, about 95% or about99%. In some embodiments, the Fah-deficient rat comprises heterozygousor homozygous insertions in the Fah gene (such as an insertion thatincludes an in-frame stop codon), with homozygous insertions beingparticularly preferred. In some embodiments, the insertion is in exon 3of Fah. In some embodiments, the Fah-deficient rat comprisesheterozygous or homozygous deletions in the Fah gene, with homozygousdeletions being particularly preferred. As one example, the deletion isin exon 3 of Fah. In another embodiment, the Fah-deficient rat comprisesone or more point mutations in the Fah gene. Examples of suitable Fahpoint mutations are known in the art (see, e.g., Aponte et al. Proc.Natl. Acad. Sci. U.S.A. 98(2):641-645, 2001).

Deplete: To reduce or remove. For example, “macrophage depletion” refersto the process of eliminating, removing, reducing or killing macrophagesin an animal. An animal that has been depleted of macrophages is notnecessarily completely devoid of macrophages but at least exhibits areduction in the number or activity of macrophages. In one embodiment,macrophage depletion results in at least a 10%, at least a 25%, at leasta 50%, at least a 75%, at least a 90% or a 100% reduction in functionalmacrophages.

Disruption: As used herein, a “disruption” in a gene refers to anyinsertion, deletion or point mutation, or any combination thereof. Insome embodiments, the disruption leads to a partial or complete loss ofexpression of mRNA and/or functional protein.

Engraft: To implant cells or tissues in an animal. As used herein,engraftment of heterologous hepatocytes in a recipient rat refers to theprocess of heterologous hepatocytes becoming implanted in the recipientrat following injection. Engrafted heterologous hepatocytes are capableof expansion in the recipient rat.

Expand: To increase in quantity. As used herein, “expanding”heterologous hepatocytes refers to the process of allowing cell divisionto occur such that the hepatocytes actively proliferate in vivo and thenumber of heterologous hepatocytes increases as compared to the originalnumber of heterologous hepatocytes transplanted into the recipient rat.

Fetus: The unborn offspring of an animal in the postembryonic period.

FK506: FK506, also known as tacrolimus or fujimycin, is animmunosuppressant drug. FK506 a 23-membered macrolide lactone firstdiscovered in the fermentation broth of a Japanese soil sample thatcontained the bacteria Streptomyces tsukubaensis. This compound is oftenused after allogeneic organ transplant to reduce the activity of thepatient's immune system and lower the risk of organ rejection. FK506reduces T-cell and interleukin-2 activity. It is also used in a topicalpreparation in the treatment of severe atopic dermatitis (eczema),severe refractory uveitis after bone marrow transplants, and the skincondition vitiligo.

Fludarabine: A purine analog that inhibits DNA synthesis. Fludarabine isoften used as a chemotherapeutic drug for the treatment of varioushematologic malignancies.

FRG KO mouse: A mutant mouse having homozygous deletions in thefumarylacetoacetate hydrolase (Fah), recombination activating gene 2(Rag2) and hemizygous or homozygous deletions of common-γ chain of theinterleukin receptor (Il2rg) x-linked gene. Also referred to asFah^(−/−)/Rag2^(−/−)/Il2rg^(−/−) or Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/y). Asused herein, homozygous deletions in the Fah, Rag2 and Il2rg genesindicates no functional FAH, RAG-2 and IL-2Rγ protein is expressed inmice comprising the mutations.

Fumarylacetoacetate hydrolase (FAH): A metabolic enzyme that catalyzesthe last step of tyrosine catabolism. Mice having a homozygous deletionof the Fah gene exhibit altered liver mRNA expression and severe liverdysfunction (Grompe et al. Genes Dev. 7:2298-2307, 1993). Pointmutations in the Fah gene have also been shown to cause hepatic failureand postnatal lethality (Aponte et al. Proc. Natl. Acad. Sci. U.S.A.98(2):641-645, 2001). Humans deficient for Fah develop the liver diseasehereditary tyrosinemia type 1 (HT1) and develop liver failure. Fahdeficiency leads to accumulation of fumarylacetoacetate, a potentoxidizing agent and this ultimately leads to cell death of hepatocytesdeficient for Fah. Thus, Fah-deficient rats can be repopulated withhepatocytes from other species, including humans. Fah genomic, mRNA andprotein sequences for a number of different species are publicallyavailable, such as in the GenBank database (see, for example, Gene ID29383 (rat Fah); Gene ID 14085 (mouse Fah); Gene ID 610140 (dog FAH);Gene ID 415482 (chicken FAH); Gene ID 100049804 (horse FAH); Gene ID712716 (rhesus macaque FAH); Gene ID 100408895 (marmoset FAH); Gene ID100589446 (gibbon FAH); Gene ID 467738 (chimpanzee FAH); and Gene ID508721 (cow FAH)).

Hepatic pathogen: Refers to any pathogen, such as a bacterial, viral orparasitic pathogen, that infects cells of the liver. In someembodiments, the hepatic pathogen is a “hepatotropic virus” (a virusthat targets the liver), such as HBV or HCV.

Hepatocellular carcinoma (HCC): HCC is a primary malignancy of the livertypically occurring in patients with inflammatory livers resulting fromviral hepatitis, liver toxins or hepatic cirrhosis.

Hepatocyte: A type of cell that makes up 70-80% of the cytoplasmic massof the liver. Hepatocytes are involved in protein synthesis, proteinstorage and transformation of carbohydrates, synthesis of cholesterol,bile salts and phospholipids, and detoxification, modification andexcretion of exogenous and endogenous substances. The hepatocyte alsoinitiates the formation and secretion of bile. Hepatocytes manufactureserum albumin, fibrinogen and the prothrombin group of clotting factorsand are the main site for the synthesis of lipoproteins, ceruloplasmin,transferrin, complement and glycoproteins. In addition, hepatocytes havethe ability to metabolize, detoxify, and inactivate exogenous compoundssuch as drugs and insecticides, and endogenous compounds such assteroids.

Hepatocyte progenitor: Any cell type capable of giving rise to ahepatocyte. Examples of hepatocyte progenitors include, but are notlimited to, embryonic stem cells (ESCs), induced pluripotent stem cells(IPSC), adult intrahepatic stem cells, mesenchymal stem cells andamniotic stem cells. In the context of the present disclosure, methodsof expanding heterologous hepatocytes in vivo include transplantingheterologous hepatocyte or hepatocyte progenitors.

Hereditary tyrosinemia type 1 (HT1): Tyrosinemia is an error ofmetabolism, usually inborn, in which the body cannot effectively breakdown the amino acid tyrosine. HT1 is the most severe form of thisdisorder and is caused by a shortage of the enzyme fumarylacetoacetatehydrolase (FAH) encoded by the gene Fah found on human chromosome number15. FAH is the last in a series of five enzymes needed to break downtyrosine. Symptoms of HT1 usually appear in the first few months of lifeand include failure to gain weight and grow at the expected rate(failure to thrive), diarrhea, vomiting, yellowing of the skin andwhites of the eyes (jaundice), cabbage-like odor, and increased tendencyto bleed (particularly nosebleeds). HT1 can lead to liver and kidneyfailure, problems affecting the nervous system, and an increased risk ofliver cancer.

Heterologous: Derived from a source other than (i.e., foreign to) thereferenced species in contrast to material derived from, naturallyassociated with, or native to, that species. In the case of the ratmodels herein, the rats can be transplanted and engrafted withhepatocytes from species other than the recipient rat, such as humans,dogs, pigs, etc.

Heterozygous: Having dissimilar alleles at corresponding chromosomalloci. For example, a rat heterozygous for a particular gene mutation hasthe mutation in one allele of the gene but not the other.

Homozygous: Generally means having identical alleles at one or moreloci. As used herein, “homozygous” for disruptions or “homozygous” for adeficiency refers to an organism having disruptions (such as a deletion,insertion or point mutation) of both alleles of a gene, as well as“compound heterozygosity” in which the organism may have disruptions ontwo unrelated alleles, which nevertheless behave like a homozygousdisruption, resulting in loss of functional gene products.

Humanized: As used herein, references to “humanized” animals (rats) orlivers refers to animals or livers transplanted with human hepatocytesin which the human hepatocytes have expanded in vivo to substantiallyrepopulate the liver of the animal with human hepatocytes.

Immunodeficient: Lacking in at least one essential function of theimmune system. As used herein, an “immunodeficient” rat is one lackingspecific components of the immune system or lacking function of specificcomponents of the immune system (such as, for example, B cells, T cellsor NK cells). In some cases, an immunodeficient rat lacks macrophages.In some embodiments, an immunodeficient rat comprises one or moregenetic alterations that prevent or inhibit the development offunctional immune cells (such as B cells, T cells or NK cells). In someembodiments, the genetic alteration is selected from the groupconsisting of recombination activating gene 1 (Rag1) deficiency,recombination activating gene 2 (Rag2) deficiency, interleukin-2receptor gamma chain (Il2rg) deficiency, the Dnapk^(−/−) (SCID)mutation, the humanized/humanized SIRP-alpha genotype, the nude ratmutation, perforin knockouts, and combinations thereof. For example,replacement of the rat Sirp-αgene with human sequence, i.e. humanization(hum) Sirp-α^(hum/hum) will block activation of rat macrophages by humancells. In some embodiments, an “immunodeficient rat” comprises one ormore of the following genetic alterations: Rag1^(−/−), Rag2^(−/−),Il2rg^(−/−), Il2rg^(−/y), SCID, SIRP-alpha genotype, perforin^(−/−),and/or nude. Immunodeficient animal strains are well known in the artand are commercially available, such as from The Jackson Laboratory (BarHarbor, Me.) or Taconic (Hudson, N.Y.). In some embodiments, the rat isa Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−)/Il1r1^(−/−) rat, aFah^(−/−)/Rag1^(−/−)/Il2rg^(−/−)/Il1r1^(−/−) rat, aFah^(−/−)/Rag2^(−/−)/Il2rg^(−/−)/Il1r1^(−/−) rat, or aFah^(−/−)/Rag1^(−/−)/Il2rg^(−/−)/Il1r1^(−/−) rat. In some embodiments,an immunodeficient rat is an animal that has been administered one ormore immunosuppressants.

Immunosuppressant: Any compound that decreases the function or activityof one or more aspects of the immune system, such as a component of thehumoral or cellular immune system or the complement system.Immunosuppressants are also referred to as “immunosuppressive agents.”Exemplary immunosuppressants include, but are not limited to: (1)antimetabolites, such as purine synthesis inhibitors (e.g., azathioprineand mycophenolic acid), pyrimidine synthesis inhibitors (e.g.,leflunomide and teriflunomide) and antifolates (e.g., methotrexate); (2)macrolides, such as FK506, cyclosporine A and pimecrolimus; (3) TNF-αinhibitors, such as thalidomide and lenalidomide; (4) IL-1 receptorantagonists, such as anakinra; (5) mammalian target of rapamycin (mTOR)inhibitors, such as rapamycin (sirolimus), deforolimus, everolimus,temsirolimus, zotarolimus and biolimus A9; (6) corticosteroids, such asprednisone; and (7) antibodies to any one of a number of cellular orserum targets. In particular embodiments of the disclosure, theimmunosuppressant is FK506, cyclosporin A, fludarabine, mycophenolate,prednisone, rapamycin or azathioprine, or combinations thereof.

Exemplary cellular targets and their respective inhibitor compoundsinclude, but are not limited to complement component 5 (e.g.,eculizumab); tumor necrosis factors (TNFs) (e.g., infliximab,adalimumab, certolizumab pegol, afelimomab and golimumab); IL-5 (e.g.,mepolizumab); IgE (e.g., omalizumab); BAYX (e.g., nerelimomab);interferon (e.g., faralimomab); IL-6 (e.g., elsilimomab); IL-12 andIL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3,otelixizumab, teplizumab, visilizumab); CD4 (e.g., clenoliximab,keliximab and zanolimumab); CD11a (e.g., efalizumab); CD18 (e.g.,erlizumab); CD20 (e.g., afutuzumab, ocrelizumab, pascolizumab); CD23(e.g., lumiliximab); CD40 (e.g., teneliximab, toralizumab);CD62L/L-selectin (e.g., aselizumab); CD80 (e.g., galiximab);CD147/basigin (e.g., gavilimomab); CD154 (e.g., ruplizumab); BLyS (e.g.,Belimumab); CTLA-4 (e.g., ipilimumab, tremelimumab); CAT (e.g.,bertilimumab, lerdelimumab, metelimumab); integrin (e.g., natalizumab);IL-6 receptor (e.g., Tocilizumab); LFA-1 (e.g., odulimomab); and IL-2receptor/CD25 (e.g., basiliximab, daclizumab, inolimomab).

Other immunosuppressive agents include zolimomab aritox, atorolimumab,cedelizumab, dorlixizumab, fontolizumab, gantenerumab, gomiliximab,maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab,siplizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab,anti-thymocyte globulin, anti-lymphocyte globulin; CTLA-4 inhibitors(e.g., abatacept, belatacept); aflibercept; alefacept; rilonacept; andTNF inhibitors (e.g., etanercept).

Immunosuppression: Refers to the act of reducing the activity orfunction of the immune system. Immunosuppression can be achieved byadministration of an immunosuppressant compound or can be the effect ofa disease or disorder (for example, immunosuppression that results fromHIV infection or due to a genetic defect). In some cases,immunosuppression occurs as the result of a genetic mutation thatprevents or inhibits the development of functional immune cells.

Induced pluripotency stem cells (iPSC): A type of pluripotent stem cellartificially derived from a non-pluripotent cell, typically an adultsomatic cell, by inducing expression of certain genes. iPSCs can bederived from any organism, such as a mammal. In some embodiments, iPSCsare produced from mice, rats, rabbits, guinea pigs, goats, pigs, cows,non-human primates or humans. Human derived iPSCs are exemplary. iPSCsare similar to ES cells in many respects, such as the expression ofcertain stem cell genes and proteins, chromatin methylation patterns,doubling time, embryoid body formation, teratoma formation, viablechimera formation, and potency and differentiability. Methods forproducing iPSCs are known in the art. For example, iPSCs are typicallyderived by transfection of certain stem cell-associated genes (such asOct-3/4 (Pouf51) and Sox2) into non-pluripotent cells, such as adultfibroblasts. Transfection can be achieved through viral vectors, such asretroviruses, lentiviruses, or adenoviruses. For example, cells can betransfected with Oct3/4, Sox2, Klf4, and c-Myc using a retroviral systemor with OCT4, SOX2, NANOG, and LIN28 using a lentiviral system. After3-4 weeks, small numbers of transfected cells begin to becomemorphologically and biochemically similar to pluripotent stem cells, andare typically isolated through morphological selection, doubling time,or through a reporter gene and antibiotic selection. In one example,iPSCs from adult human cells are generated by the method of Yu et al.(Science 318(5854):1224, 2007) or Takahashi et al. (Cell 131(5):861-72,2007). iPSCs are also known as iPS cells.

Infectious load: Refers to the quantity of a particular pathogen in asubject or in a sample from the subject. Infectious load can be measuredusing any one of a number of methods known in the art. The selectedmethod will vary depending on the type of pathogen to be detected andthe reagents available to detect the pathogen. Infectious load can alsobe measured, for example, by determining the titer of the pathogen, themethod for which will vary depending on the pathogen to be detected. Forexample, the titer of some viruses can be quantified by performing aplaque assay. In some examples, infectious load is measured byquantifying the amount of a pathogen-specific antigen in a sample. Inother examples, infectious load is measured by quantifying the amount ofa pathogen-specific nucleic acid molecule in a sample. Quantifyingencompasses determining a numerical value or can be a relative value.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or extracted away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal nucleic acids, adjacentsequences, proteins and organelles. Nucleic acids and proteins that havebeen “isolated” include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids. In either case, “isolated” nucleic acidsand/or proteins result in new chemical entities not found in nature.

Mammalian target of rapamycin (mTOR) inhibitor: A molecule that inhibitsexpression or activity of mTOR. mTOR inhibitors include, but are notlimited to small molecule, antibody, peptide and nucleic acidinhibitors. For example, an mTOR inhibitor can be a molecule thatinhibits the kinase activity of mTOR or inhibits binding of mTOR to aligand. Inhibitors of mTOR also include molecules that down-regulateexpression of mTOR. A number of mTOR inhibitors are known in the art,including rapamycin (sirolimus).

Mycophenolate: An immunosuppressant typically used to prevent rejectionof allogeneic transplants. This drug is generally administered orally orintravenously. Mycophenolate is derived from the fungus Penicilliumstoloniferum. Mycophenolate mofetil, the pro-drug form, is metabolizedin the liver to the active moiety mycophenolic acid. It inhibits inosinemonophosphate dehydrogenase, the enzyme that controls the rate ofsynthesis of guanine monophosphate in the de novo pathway of purinesynthesis used in the proliferation of B and T lymphocytes. Mycophenolicacid is commonly marketed under the trade names CellCept™ (mycophenolatemofetil; Roche) and Myfortic™ (mycophenolate sodium; Novartis).

NTBC (2-nitro-4-trifluoro-methyl-benzoyl)-1,3 cyclohexanedione, alsoknown as nitisinone): An inhibitor of 4-hydroxy-phenylpyruvatedioxygenase (HPPD). HPPD catalyzes the conversion of4-hydroxyphenylpyruvate to homogentisate, the second step in tyrosinecatabolism. Treatment with NTBC blocks the tyrosine catabolism pathwayat this step and prevents the accumulation of succinylacetone, apathognomonic metabolite that accumulates in Fah-deficient humans andanimals.

Nude rat: Refers to a rat strain with a genetic mutation that causes adeteriorated or absent thymus, resulting in an inhibited immune systemdue to a greatly reduced number of T cells.

Prednisone: A synthetic corticosteroid that is an effectiveimmunosuppressant. It is often used to treat certain inflammatorydiseases, autoimmune diseases and cancers as well as treat or preventorgan transplant rejection. Prednisone is usually taken orally but canbe delivered by intramuscular injection or intravenous injection. It isa prodrug that is converted by the liver into prednisolone, which is theactive drug and also a steroid.

Rapamycin: A compound with known immunosuppressive andanti-proliferative properties. Rapamycin, also known as sirolimus, is amacrolide that was first discovered as a product of the bacteriumStreptomyces hygroscopicus. Rapamycin binds and inhibits the activity ofmTOR.

Recipient: As used herein, a “recipient rat” is a rat that has beeninjected with heterologous hepatocytes as described herein. Typically, aportion (the percentage can vary) of the heterologous hepatocytesengraft in the recipient rat. In some embodiments, the recipient rat isimmunosuppressed or immunodeficient. In some embodiments, the recipientrat is Fah-deficient.

Recombination activating gene 1 (Rag1): A gene involved in activation ofimmunoglobulin V(D)J recombination. The RAG1 protein is involved inrecognition of the DNA substrate, but stable binding and cleavageactivity also requires RAG2. Rag-1-deficient rats have no mature B and Tlymphocytes.

Recombination activating gene 2 (Rag2): A gene involved in recombinationof immunoglobulin and T cell receptor loci. Rats deficient in the Rag2gene are unable to undergo V(D)J recombination, resulting in a completeloss of functional T cells and B cells (Shinkai et al. Cell 68:855-867,1992).

Serial transplantation: The process for expanding heterologoushepatocytes in vivo in which hepatocytes expanded in a first animal arecollected and transplanted, such as by injection, into a secondaryanimal for further expansion. Serial transplantation can further includetertiary, quaternary or additional animals.

Somatic cell nuclear transfer (SCNT): A laboratory technique forcreating a clonal embryo, using an ovum with a donor nucleus. In SCNT,the nucleus of a somatic cell is removed and the rest of the celldiscarded. At the same time, the nucleus of an egg cell is removed. Thenucleus of the somatic cell is then inserted into the enucleated eggcell. After being inserted into the egg, the somatic cell nucleus isreprogrammed by the host cell. The egg, now containing the nucleus of asomatic cell, is stimulated with a shock and will begin to divide. Aftermany mitotic divisions in culture, this single cell forms a blastocystwith almost identical DNA to the original organism.

Stem cell: A cell having the unique capacity to produce unaltereddaughter cells (self-renewal; cell division produces at least onedaughter cell that is identical to the parent cell) and to give rise tospecialized cell types (potency). Stem cells include, but are notlimited to, embryonic stem (ES) cells, embryonic germ (EG) cells,germline stem (GS) cells, human mesenchymal stem cells (hMSCs), adiposetissue-derived stem cells (ADSCs), multipotent adult progenitor cells(MAPCs), multipotent adult germline stem cells (maGSCs) and unrestrictedsomatic stem cell (USSCs). The role of stem cells in vivo is to replacecells that are destroyed during the normal life of an animal. Generally,stem cells can divide without limit. After division, the stem cell mayremain as a stem cell, become a precursor cell, or proceed to terminaldifferentiation. A precursor cell is a cell that can generate a fullydifferentiated functional cell of at least one given cell type.Generally, precursor cells can divide. After division, a precursor cellcan remain a precursor cell, or may proceed to terminal differentiation.In one embodiment, the stem cells give rise to hepatocytes.

Therapeutic agent: A chemical compound, small molecule, or othercomposition, such as an antisense compound, antibody, proteaseinhibitor, hormone, chemokine or cytokine, capable of inducing a desiredtherapeutic or prophylactic effect when properly administered to asubject. As used herein, a “candidate agent” is a compound selected forscreening to determine if it can function as a therapeutic agent for aparticular disease or disorder.

Titer: In the context of the present disclosure, titer refers to theamount of a particular pathogen in a sample.

Tolerance: A state of unresponsiveness to a specific antigen or group ofantigens to which a subject (such as a human or animal) is normallyresponsive. Immune tolerance is achieved under conditions that suppressthe immune reaction and is not just the absence of an immune response.Immune tolerance can result from a number of causes including priorcontact with the same antigen in fetal life or in the newborn periodwhen the immune system is not yet mature; prior contact with the antigenin extremely high or low doses; exposure to radiation, chemotherapydrugs, or other agents that impair the immune system; heritable diseasesof the immune system; and acquired diseases of the immune system.

Toxin: In the context of the present disclosure, “toxin” refers to anypoisonous substance, including any chemical toxin or biological toxin.

Transgene: An exogenous nucleic acid sequence introduced into a cell orthe genome of an organism.

Transplant or transplanting: Refers to the process of grafting an organ,tissue or cells from one subject (e.g., human or animal) to anothersubject, or to another region of the same subject.

Urokinase: Also called urokinase-type Plasminogen Activator (uPA),urokinase is a serine protease. Urokinase was originally isolated fromhuman urine, but is present in several physiological locations, such asthe blood stream and the extracellular matrix. The primary physiologicalsubstrate is plasminogen, which is an inactive zymogen form of theserine protease plasmin. Activation of plasmin triggers a proteolyticcascade which, depending on the physiological environment, participatesin thrombolysis or extracellular matrix degradation. In one embodimentof the methods provided herein, urokinase is administered to a recipientrat prior to hepatocyte injection. In some embodiments, urokinase ishuman urokinase. In some embodiments, the human urokinase is thesecreted form of urokinase. In some embodiments, the human urokinase isa modified, non-secreted form of urokinase (see U.S. Pat. No.5,980,886).

Vector: A nucleic acid molecule allowing insertion of foreign nucleicacid without disrupting the ability of the vector to replicate and/orintegrate in a host cell. A vector can include nucleic acid sequencesthat permit it to replicate in a host cell, such as an origin ofreplication. A vector can also include one or more selectable markergenes and other genetic elements. An integrating vector is capable ofintegrating itself into a host nucleic acid. An expression vector is avector that contains the necessary regulatory sequences to allowtranscription and translation of inserted gene or genes.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the term “and/or” when used in a list oftwo or more items, means that any one of the listed items can beemployed by itself or any combination of two or more of the listed itemscan be employed. For example, if a composition comprises or excludescomponents A, B, and/or C, the composition can contain or exclude Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

II. Overview of Several Embodiments

Described herein are genetically modified rats (Rattus sp.), which haveutility for a variety of purposes, including for the expansion ofhepatocytes from other species (particularly humans), and as animalmodels of liver diseases and normal liver pathology, includingintermediate metabolism, bile acid production, small moleculetransporters, assessment of biologics, drug metabolism,pharmacokinetics, as well as cirrhosis, hepatocellular carcinoma,hepatic infection, hepatic toxicity, gene therapy, liver regeneration,alcoholic liver disease, fatty liver disease, metabolic liver disease,and the like. The term “rat,” as used herein, is specific to the genusRattus, and expressly does not encompass mice species (Mus sp.).

In one or more embodiments, the rats are Fah-deficient, and preferablycomprise a genome that is homozygous for a disruption in the Fah gene,such that the disruption results in loss of expression of functional FAHprotein resulting in decreased liver function. The Fah gene disruptionneed not result in a complete loss of expression of functional FAHprotein. In some examples, loss or decrease of expression, activity, orfunction, of functional FAH protein is loss of expression of about 80%,about 90%, about 95% or about 99% of functional protein. The Fah genedisruption can be any modification that results in a significantdiminishment (reduction) or complete loss of expression of functionalFAH protein. In some embodiments, the disruption is an insertion, adeletion, or one or more point mutations in the Fah gene, or anycombination thereof. For example, the disruption can be an insertionthat includes an in-frame stop codon. The insertion can also includeadditional nucleic acid sequences, such as nucleic acid encoding aselectable marker. In particular examples, the Fah-deficient rat ishomozygous for disruptions in exon 3 of the Fah gene. Further providedare isolated rat embryonic stem (ES) cells comprising a disruption inthe Fah gene.

Also provided herein is a method of producing an Fah-deficient rat cell(such as an embryonic stem cell) by transfecting the rat cell with anAAV-DJ targeting vector, wherein the targeting vector comprises atargeting cassette comprising 5′ and 3′ homology arms that target aportion of the rat Fah gene (such as exon 3 of the Fah gene) and whichflank a nucleic acid sequence (such as nucleic acid sequence encoding anantibiotic resistance gene), such that following transfection of thecell with the targeting vector, homologous recombination occurs,replacing the targeted portion of the rat Fah gene with the targetingcassette, thereby producing an Fah-deficient rat cell.

In some embodiments, an immunodeficient rat is provided. Theimmunodeficiency of the mouse can be due to a genetic alteration,immunosuppression, or a combination thereof. In one or more embodiments,the rat lacks functional T cells, B cells, and/or NK cells.

In some embodiments, the rat is immunosuppressed. In some examples,immunosuppression is the result of administration of one or moreimmunosuppressive agents. Any suitable immunosuppressive agent or agentseffective for achieving immunosuppression in the rat can be used.Examples of immunosuppressive agents include, but are not limited to,FK506, cyclosporin A, fludarabine, mycophenolate, prednisone, rapamycinand azathioprine. Combinations of immunosuppressive agents can also beadministered.

In other embodiments, the immunodeficiency is the result of one or moregenetic alterations that result in a lack of a specific component of theimmune system, or a lack of functionality of a specific component of theimmune system (such as a lack of functional B, T and/or NK cells). Insome examples, the one or more genetic alterations comprise a geneticalteration of the Rag2 gene or a genetic alteration of the Il2rg genesuch that the genetic alteration results in loss of expression offunctional RAG-2 protein or IL-2Rγ protein. In one example, the one ormore genetic alterations comprise a genetic alteration of the Rag2 geneand a genetic alteration of the Il2rg gene. In some cases, the geneticalteration comprises homozygous deletions in the Rag2 gene or the Il2rggene. Such deletions, in combination with the Fah-deficiency result inan FRG KO rat. In other examples, the genetic alteration is SCID, NOD,or nude. Specific cells of the immune system (such as macrophages or NKcells) can also be depleted. Methods of depleting particular cell typesare known in the art.

In one or more embodiments, the rat comprises heterologous hepatocytes,such as human hepatocytes. It will be appreciated that a significantadvantage of the invention is that the heterologous hepatocytes have notjust been transplanted/engrafted in the rat, but expanded (i.e.,actively proliferated) in the rat. In other words, various embodimentsof the invention are concerned with rats having humanized livers.

Methods of expanding heterologous hepatocytes in vivo are also provided.In some embodiments, the method includes transplanting heterologoushepatocytes (or hepatocyte progenitors) into an Fah-deficient rat andallowing the heterologous hepatocytes to expand. In other embodiments,the method includes transplanting heterologous hepatocytes into animmunodeficient rat and allowing the heterologous hepatocytes to expand.In yet other embodiments, the method includes transplanting heterologoushepatocytes into an Fah-deficient and immunodeficient rat and allowingthe heterologous hepatocytes to expand. In other embodiments, the methodincludes transplanting heterologous hepatocytes into a rat with ahepatic deficiency, such as decreased expression, activity, or functionof an enzyme in the tyrosine catabolic pathway, and allowing theheterologous hepatocytes to expand.

In some embodiments, the heterologous hepatocytes are transplanted byinjection into the hepatic artery, spleen, portal vein, peritonealcavity, hepatic tissue mass, or lymphatic system of the rat. In someembodiments, the heterologous hepatocytes (or hepatocyte progenitors)transplanted into the rat are isolated human hepatocytes (or hepatocyteprogenitors). In some embodiments, the heterologous hepatocytes aretransplanted as part of a liver tissue graft. The isolated heterologoushepatocytes can be obtained from any one of a number of differentmammals as well as different sources. Mammalian hepatocytes can beobtained from dogs, pigs, horses, rabbits, mice, marmoset, woodchuck,non-human primates, and humans. In one embodiment, the heterologoushepatocytes are isolated from the liver of a human or non-human organdonor. In another embodiment, the heterologous hepatocytes are isolatedfrom a surgical resection. In another embodiment, the heterologoushepatocytes are derived from a stem cell, such as an embryonic stemcell, an induced pluripotency stem cell, a mesenchymal-derived stemcell, an adipose tissue-derived stem cell, a multipotent adultprogenitor cell, an unrestricted somatic stem cell or tissue-specificliver stem cell, which can be found in the liver itself, the gallbladder, the intestine, the pancreas, or salivary glands. In anotherembodiment, the heterologous hepatocytes are derived from monocytes oramniocytes, thus a stem cell or progenitor cell is obtained in vitro toproduce hepatocytes. In another embodiment, the heterologous hepatocytesare derived by reprogramming a distinct cell lineage such as a skinfibroblast, keratinocyte or lymphocyte. In another embodiment, theheterologous hepatocytes have been cryopreserved prior to injection(i.e., are from cryopreserved lots which are thawed). In someembodiments, human hepatocytes can be isolated from humanized mice,pigs, or other rats, as discussed in more detail herein.

In some embodiments, the one or more immunosuppressive agents (discussedabove) are administered to the rat at least about 2 days prior toheterologous hepatocyte transplantation. Immunosuppression may becontinued after transplantation for a period of time, for example for aportion of or the entire life of the rat.

It will be appreciated that various models of liver injury may be usedin the rat to facilitate hepatocyte engraftment and expansion,including, without limitation, inducible injury and/or geneticmodifications (e.g., Fah disruption, uPA, TK-NOG (Washburn et al.,Gastroenterology, 140(4):1334-44, 2011), albumin AFC8, albumindiphtheria toxin, Wilson's Disease, and the like). Combinations of liverinjury techniques may also be used. In some embodiments, the rat isadministered a vector encoding a urokinase gene prior to injection ofthe heterologous hepatocytes. In one embodiment, the urokinase gene ishuman urokinase. Wild-type urokinase is a secreted protein. Thus, insome embodiments, the human urokinase is a secreted form of urokinase(Nagai et al., Gene 36:183-188, 1985). In some embodiments, the humanurokinase is a modified, non-secreted form of urokinase. For example,Lieber et al. (Proc. Natl. Acad. Sci. 92:6210-6214, 1995) describenon-secreted forms of urokinase generated by inserting a sequenceencoding an endoplasmic reticulum retention signal at the carboxylterminus of urokinase, or by replacing the pre-uPA signal peptide withthe amino-terminal RR-retention signal (Strubin et al., Cell 47:619-625,1986; Schutze et al., EMBO J. 13:1696-1705, 1994) and the transmembraneanchor separated by a spacer peptide from the membrane II protein Iip33(Strubin et al., Cell 47:619-625, 1986). Non-secreted forms of urokinaseare also described in U.S. Pat. No. 5,980,886.

The vector encoding urokinase can be any type of vector suitable fordelivery to the rat and capable of expressing the urokinase gene. Suchvectors include viral vectors or plasmid vectors. In one embodiment, thevector is an adenovirus vector. In another embodiment, the vector is anAAV vector. The vector encoding urokinase can be administered by anysuitable means known in the art. In one embodiment, the vector isadministered intravenously. In one aspect, the vector is administered byretroorbital injection. The vector encoding urokinase can beadministered any time prior to injection of the heterologoushepatocytes. Typically, the vector is administered to allow sufficienttime for urokinase to be expressed. In one embodiment, the vector isadministered 24 to 48 hours prior to hepatocyte injection.

The length of time for hepatocyte expansion can vary and will depend ona variety of factors, including the number of hepatocytes originallytransplanted, the number of heterologous hepatocytes desired followingexpansion and/or the desired degree of liver repopulation with theheterologous hepatocytes. In some cases, these factors will be dictatedby the desired use of the hepatocytes or the desired use of the ratengrafted with the heterologous hepatocytes. In some embodiments, theheterologous hepatocytes are expanded in the rat for at least about 3days, at least about 5 days, at least about 7 days, at least about 2weeks, or at least about 4 weeks, at least about 5 weeks, at least about6 weeks, at least about 2 months, at least about 3 months, at leastabout 4 months, at least about 5 months, at least about 6 months, atleast about 7 months, at least about 8 months, at least about 9 months,at least about 10 months or at least about 11 months. In particularexamples, the heterologous hepatocytes are expanded in the rat for atleast 7 days. In other examples, the heterologous hepatocytes areexpanded in the rat for at least 6 months. In some examples, theheterologous hepatocytes are expanded in the rat no more than 12 months.

In some embodiments, the rats with a hepatic deficiency can beadministered an agent that inhibits, delays, avoids or prevents thedevelopment of liver disease in the rat. Administration of such an agentavoids liver dysfunction and/or death of the rat prior to repopulationof the rat with healthy (e.g., FAH-expressing) hepatocytes. The agentcan be any compound or composition known in the art to inhibit liverdisease. One such agent is 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1,3cyclohexanedione (NTBC), but other pharmacologic inhibitors ofphenylpyruvate dioxygenase, such as methyl-NTBC can be used. NTBC (oranother compound with a liver protective effect) is administered toregulate the development of liver disease in the Fah-deficient rat. Thedose, dosing schedule and method of administration can be adjusted asneeded to avoid liver dysfunction in the Fah-deficient rat. In someembodiments, the Fah-deficient rat is further administered NTBC for atleast two days, at least three days, at least four days, at least fivedays or at least six days following hepatocyte transplantation. In someembodiments, the Fah-deficient rat is further administered NTBC for atleast about one week, at least about two weeks, at least about threeweeks, at least about four weeks, at least about one month, at leastabout two months, at least about three months, at least about fourmonths, at least about five months, or at least about six months. Insome embodiments, the NTBC (or another compound with a liver protectiveeffect) is withdrawn at about two days, about three days, about fourdays, about five days, about six days or about seven days followinghepatocyte transplantation.

The dose of NTBC administered to the Fah-deficient rat can vary. In someembodiments, the dose is about 0.5 mg/kg to about 30 mg/kg per day,preferably from about 1 mg/kg to about 25 mg/kg, more preferably fromabout 10 mg/kg per day to about 20 mg/kg per day, and even morepreferably about 20 mg/kg per day. NTBC can be administered by anysuitable means, such as, but limited to, in the drinking water, in thefood or by injection. In one embodiment, the concentration of NTBCadministered in the drinking water is about 1 to about 30 mg/L,preferably from about 10 to about 25 mg/L, more preferably from about 15to about 20 mg/L, and even more preferably about 20 mg/L.

In some embodiments, the method further includes collecting the expandedheterologous hepatocytes from the rat. Further provided is an expandedheterologous hepatocyte isolated from the liver of the rat.

Further provided herein is a method of serial transplantation ofheterologous hepatocytes (or hepatocytes from another species) in therecipient rat. The method comprises collecting the expanded heterologoushepatocytes from a first recipient rat and further expanding thehepatocytes in a second, third, fourth or additional recipient rat. Theexpanded hepatocytes can be collected from the rat using any one of anumber of techniques. For example, the hepatocytes can be collected byenzymatic digestion of the rat liver, followed by gentle mincing,filtration, and centrifugation. Furthermore, the hepatocytes can beseparated from other cell types, tissue and/or debris using well-knownmethods, such as by using an antibody that specifically recognizes thecell type of the engrafted hepatocyte species. Such antibodies include,but are not limited to an antibody that specifically binds to a class Imajor histocompatibility antigen, such as anti-human HLA-A,B,C (Markuset al. Cell Transplantation 6:455-462, 1997). Antibody bound hepatocytescan then be separated by panning (which utilizes a monoclonal antibodyattached to a solid matrix), fluorescence activated cell sorting (FACS),magnetic bead separation or the like. Alternative methods of collectinghepatocytes are well known in the art.

Further provided is a method of assessing the effect of an exogenousagent (xenobiotic) on hepatocytes, such as human hepatocytes, in vivo.In some embodiments, the method includes administering the exogenousagent to a rat model described herein which has been transplanted withheterologous hepatocytes and measuring at least one marker of liverfunction in the rat. In some embodiments, the at least one marker ofliver function is selected from AST, ALT, bilirubin, alkalinephosphatase and albumin, and wherein an increase in AST, ALT, bilirubinor alkaline phosphatase, or a decrease in albumin in the rat relative tothe rat prior to administration of the exogenous agent, indicates theexogenous agent is toxic. In some embodiments, the exogenous agent is aknown or suspected toxin.

III. Animal Models and Uses Thereof

According to one embodiment described herein is a method for generationof Fah-deficient rats by homologous recombination using an AAV targetingconstruct in rat embryonic stem (ES) cells. The examples describe thegeneration of an AAV targeting construct containing aneomycin-resistance cassette and 5′ and 3′ homology arms that target theconstruct for insertion in exon 3 of the rat Fah gene. Rat ES cells wereinfected with the AAV targeting vector and selected for neomycinresistance to isolate individual cell clones with integrated vector. TheFAH knockout (KO) ES cells can be injected into rat blastocysts andtransferred to E3.5 pseudo-pregnant SD rats. The progeny from these ratscan be genotyped and chimeric rats can be bred to generate heterozygotemales and females that can be bred to generate homozygote FAH KO rats.This disclosure is not limited to one particular method of generating anFah-deficient rat, but encompasses any method known in the art toproduce an Fah-deficient rat, including, without limitation, TALEN, zincfinger nucleases, meganucleases, SCNT, and the like.

In one or more embodiments, additional metabolic enzymes in the tyrosinecatabolic pathway can be targeted in addition to or in lieu of Fah togenerate animal models with severe liver dysfunction (“hepaticdeficiency”). For example, genes encoding for tyrosine aminotransferase,4-hydroxy-phenylpyruvate dioxygenase, homogentisate 1,2-dioxygenase,and/or maleylacetoacetate isomerase can be targeted for disruption usingmethods similar to those described herein. In the absence of treatment,rats deficient (i.e., have decreased expression, activity, or function)in one or more of the above enzymes will develop a liver dysfunctionsuch as tyrosinemia (I, II, III), hawkinsinuria, alkaptonuria, and thelike, which ultimately results in death of endogenous hepatocytes. Thus,as with Fah-deficient models, such rats can also be repopulated withhepatocytes from other species, including humans.

Also disclosed herein is the engraftment of heterologous hepatocytes inlivers of immunodeficient rats. Accordingly, the use of immunodeficientrats (in the absence of Fah-deficiency) is contemplated herein forexpansion of heterologous hepatocytes. Humanized rats (i.e. rats havinglivers reconstituted with human hepatocytes) can also be used, forexample, as a source of hepatocytes for human liver reconstitution aswell as for pharmacology, toxicology, and gene therapy studies, asdiscussed below.

In some cases, prior to transplantation with heterologous hepatocytes,acute liver damage will be induced in the rats using any suitable modelof liver injury including uPa, TK, selective embolism, transientischemia, retrorsine, monocrotoline, irradiation with gamma rays, carbontetrachloride, and the like. In some embodiments liver damage is inducedby administering a recombinant adenovirus expressing the urokinaseplasminogen-like enzyme. After administration of urokinase (such as 24hours later), the heterologous hepatocytes can be delivered viaintra-splenic injection where the hepatocytes will travel through thevasculature to reach the liver. In addition, immune suppression drugscan optionally be given to the rats before, during and after thetransplant to eliminate the host versus graft response in the rat fromthe xenografted heterologous hepatocytes. By cycling the rats off NTBCfor defined periods of time, the rat cells become quiescent and theengrafted cells will have a proliferative advantage leading toreplacement of endogenous rat hepatocytes with heterologous hepatocytes.In the case of human hepatocytes, this generates rats with high levelsof humanized livers. Heterologous hepatocyte repopulation levels can bedetermined through quantitation of human serum albumin levels correlatedwith immunohistochemistry of liver sections from transplanted rats.

A. Expansion of Hepatocytes and their Medical Use

The present disclosure contemplates the use of heterologous hepatocytesexpanded in and collected from a recipient rat as a source ofhepatocytes for liver reconstitution in a subject in need of suchtherapy. Reconstitution of liver tissue in a patient by the introductionof hepatocytes is a potential therapeutic option for patients with acuteliver failure, either as a temporary treatment in anticipation of livertransplant or as a definitive treatment for patients with isolatedmetabolic deficiencies (Bumgardner et al. Transplantation 65: 53-61,1998). Hepatocyte reconstitution may be used, for example, to introducegenetically modified hepatocytes for gene therapy or to replacehepatocytes lost as a result of disease, physical or chemical injury, ormalignancy (U.S. Pat. No. 6,995,299). For example, use of transfectedhepatocytes in gene therapy of a patient suffering from familialhypercholesterolemia has been reported (Grossman et al. Nat. Genet. 6:335, 1994). In addition, expanded human hepatocytes can be used topopulate artificial liver assist devices. Particular methods oftransplanting and expanding heterologous hepatocytes in rats, as wellmedical uses of the expanded heterologous hepatocytes, are described ingreater detail below.

1. Pre-Immune Fetal Transplantation of Hepatocytes

One method disclosed herein for expanding heterologous hepatocytes inthe rats includes transplanting heterologous hepatocytes and/orhepatocyte progenitors into fetuses. In one or more embodiments, thedisclosed method for pre-immune hepatocyte transplantation in ratsincludes breeding Fah-deficient rats to each other to generate onlyhomozygous Fah-knockout offspring. At approximately day 9-15 ofgestation (e.g., day 12), the Fah-deficient rat fetus is surgicallyexternalized and injected with heterologous hepatocytes via theumbilical vein or directly into the fetal liver. This method would alsowork with genetically modified immunodeficient rat fetuses, or ratfetuses having another hepatic deficiency.

The number of heterologous hepatocytes injected into the rat can varyand will depend on the desired use, route of delivery and other factors.In some embodiments, the fetus is injected with from about 50,000 toabout 1×10⁸, such as about 500,000 to about 1×10⁷, heterologoushepatocytes. In some examples, the fetus is injected with about 1×10⁶ toabout 1×10⁸, such as about 1×10⁷, heterologous hepatocytes. In onenon-limiting example, the fetus is injected with approximately 1×10⁷heterologous hepatocytes directly into the fetal liver. In otherexamples, the number of heterologous hepatocytes injected is about100,000 to about 500,000, such as about 300,000. Because of the immaturenature of the immune system during fetal development, the fetus willdevelop immunological tolerance to heterologous hepatocytes. Thus, it isnot necessary to treat a rat that has been transplanted withheterologous hepatocytes during fetal development with immunosuppressiveagents.

In one or more embodiments, the pregnant rat is maintained on aneffective amount of a pharmacologic inhibitor of phenylpyruvatedioxygenase, such as NTBC or another compound, to avoid liverdysfunction or failure in the Fah-deficient fetus throughout pregnancy.The dose of pharmacologic inhibitor of phenylpyruvate dioxygenase canvary. Exemplary dosages are discussed above. The dose of NTBC can bemodified as needed to avoid liver dysfunction in the Fah-deficient rats.After birth, the Fah-deficient rat engrafted with heterologoushepatocytes will no longer be administered NTBC to permit expansion ofthe heterologous hepatocytes. In some examples, the Fah-deficient ratwill no longer be administered NTBC from immediately after birth. Inother examples, the dose of NTBC is gradually reduced over time, such asover about 1 to 6 days, such as 1 to 6 days from the date of birth.

2. Post-Natal Transplantation of Hepatocytes

A second method of expanding heterologous hepatocytes in rat modelsdescribed herein includes post-natal transplantation of the heterologoushepatocytes and/or hepatocyte progenitors into the rats. In someembodiments, the rats are transplanted shortly after birth, such aswithin 2 days or within a week of birth. In other embodiments, olderrats, including adult rats, are transplanted. The heterologoushepatocytes are generally transplanted via the hepatic artery,intrasplenic injection or portal vein. In some embodiments, theFah-deficient rats are maintained on a pharmacologic inhibitor ofphenylpyruvate dioxygenase, such as NTBC or methyl-NTBC, to inhibit oravoid liver dysfunction. Prior to transplantation of the heterologoushepatocytes, the rats can be treated with one or more immunosuppressiveagents to prevent rejection of the heterologous hepatocytes. Typically,the one or more immunosuppressive agents are administered about two daysprior to heterologous hepatocyte transplantation; however, the timingfor initiating treatment with the immunosuppressive agents can vary ifnecessary in order to achieve optimal results. Administration of theimmunosuppressive agents will typically continue indefinitely in anamount effective to avoid rejection of the heterologous hepatocytes.

Once transplantation of the heterologous hepatocytes has been completed,the Fah-deficient rats are no longer administered NTBC to allow forexpansion of the heterologous hepatocytes. The presence of theheterologous hepatocytes (which are not deficient for Fah) allows therats to remain healthy in the absence of NTBC. In some cases, treatmentwith NTBC is stopped immediately after hepatocyte transplantation. Inother cases, NTBC is gradually reduced over time, such as over about oneto about six days. In some embodiments, the dose of NTBC whenadministered to the Fah-deficient rats, is about 0.2 mg/kg to about 2.0mg/kg per day. In some examples, the dose of NTBC is about 1.0 mg/kg perday. The dose of NTBC can be modified as needed to avoid liverdysfunction in the Fah-deficient rats.

B. Sources of Heterologous Hepatocytes

Any suitable source of heterologous hepatocytes or hepatocyteprecursors/progenitors can be used in the disclosed methods fortransplantation in rats. For example, human hepatocytes can be derivedfrom cadaveric donors or liver resections from various species ofmammals, or can be obtained from commercial sources. Human hepatocytescan also be obtained from previously engrafted animals (i.e., humanizedanimals whose livers have been previously repopulated with humanhepatocytes, such as the humanized FRG KO mice disclosed in WO2008/151283 and WO 2010/127275). Methods of isolating human hepatocytesfrom such animals are described herein. It is anticipated that it willbe possible to transplant rats with heterologous hepatocytes from donorsof all ages or with cryopreserved hepatocytes. There is often a delay(typically 1 to 2 days) between isolation of human hepatocytes andtransplantation, which can result in poor viability of the hepatocytes.However, the rat systems described herein can serve as a means ofexpanding human hepatocytes even when the number of hepatocytes islimited in number.

Methods of isolating hepatocytes are well known in the art. For example,methods of isolating hepatocytes from organ donors or liver resectionsare described in WO 2004/009766 and WO 2005/028640 and U.S. Pat. Nos.6,995,299 and 6,509,514. Hepatocytes can be obtained from a liver biopsytaken percutaneously or via abdominal surgery. Heterologous hepatocytesfor transplantation into a recipient rat can be isolated from mammalianliver tissue by any convenient method known in the art. Liver tissue canbe dissociated mechanically or enzymatically to provide a suspension ofsingle cells, or fragments of intact hepatic tissue may be used. Forexample, the hepatocytes can be isolated from donor tissue by routinecollagenase perfusion (Ryan et al. Meth. Cell Biol. 13:29, 1976)followed by low-speed centrifugation. Hepatocytes can be furtherpurified by filtering through a stainless steel mesh, followed bydensity-gradient centrifugation. Alternatively, other methods forenriching for hepatocytes can be used, such as, for example,fluorescence activated cell sorting, panning, magnetic bead separation,elutriation within a centrifugal field, or any other method well knownin the art. Similar hepatocyte isolation methods can be used to collectexpanded heterologous hepatocytes from a recipient rat liver.

Alternatively, heterologous hepatocytes can be prepared using thetechnique described by Guguen-Guillouzo et al. (Cell Biol. Int. Rep.6:625-628, 1982). Briefly, a liver or portion thereof is isolated and acannula is introduced into the portal vein or a portal branch. The livertissue is then perfused, via the cannula, with a calcium-free bufferfollowed by an enzymatic solution containing collagenase (such as about0.025% collagenase) in calcium chloride solution (such as about 0.075%calcium chloride) in HEPES buffer at a flow rate of between 30 and 70milliliters per minute at 37° C. The perfused liver tissue is mincedinto small (such as about 1 cubic millimeter) pieces. The enzymaticdigestion is continued in the same buffer as described above for about10-20 minutes with gentle stirring at 37° C. to produce a cellsuspension, and the released hepatocytes are collected by filtering thecell suspension through a 60-80 micrometer nylon mesh. The collectedhepatocytes can then be washed in cold HEPES buffer at pH 7.0 using slowcentrifugation to remove collagenase and cell debris. Non-parenchymalcells may be removed by metrizamide gradient centrifugation (see U.S.Pat. No. 6,995,299).

Hepatocytes can be obtained from fresh tissue (such as tissue obtainedwithin hours of death) or freshly frozen tissue (such as fresh tissuefrozen and maintained at or below about 0° C.). For some applications,it is preferred that the heterologous tissue has no detectablepathogens, is normal in morphology and histology, and is essentiallydisease-free. The hepatocytes used for engraftment can be recentlyisolated, such as within a few hours, or can be transplanted afterlonger periods of time if the cells are maintained in appropriatestorage media. One such media is VIASPAN™ (a universal aortic flush andcold storage solution for the preservation of intra-abdominal organs;also referred to as University of Wisconsin solution, or UW).Hepatocytes also can be cryopreserved prior to transplantation. Methodsof cryopreserving hepatocytes are well known in the art and aredescribed, for example, in U.S. Pat. No. 6,136,525.

In addition to obtaining heterologous hepatocytes from organ donors orliver resections, the cells used for engraftment can be mammalian stemcells, such as human stem cells, or hepatocyte precursor cells that,following transplantation into the recipient rat, develop ordifferentiate into hepatocytes capable of expansion. Human cells with EScell properties have been isolated from the inner blastocyst cell mass(Thomson et al., Science 282:1145-1147, 1998) and developing germ cells(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726-13731, 1998), andhuman embryonic stem cells have been produced (see U.S. Pat. No.6,200,806). As disclosed in U.S. Pat. No. 6,200,806, ES cells can beproduced from human and non-human primates. Induced pluripotent stem(iPS) cells induced from human and non-human primate cells can also beobtained (see, for example, Yu et al., Science 318(5858):1917-1920,2007; Takahashi et al., Cell 131(5):861-872, 2007; Liu et al., CellStein Cell 3(6):587-590, 2008). Generally, primate ES cells are isolatedon a confluent layer of murine embryonic fibroblast in the presence ofES cell medium. ES cell medium generally consists of 80% Dulbecco'smodified Eagle's medium (DMEM; no pyruvate, high glucose formulation,Gibco BRL), with 20% fetal bovine serum (FBS; Hyclone), 0.1 mMβ-mercaptoethanol (Sigma), and 1% non-essential amino acid stock (GibcoBRL). Distinguishing features of ES cells, as compared to the committed“multipotential” stem cells present in adults, include the capacity ofES cells to maintain an undifferentiated state indefinitely in culture,and the potential that ES cells have to develop into every differentcell type. Human ES (hES) cells express SSEA-4, a glycolipid cellsurface antigen recognized by a specific monoclonal antibody (see, forexample, Amit et al., Devel. Biol. 227:271-278, 2000). In one or moreembodiments, the hepatocytes can also be from a source other than ESCs.

Human hepatocytes derived from human mesenchymal stem cells (hMSCs) canalso be used in the methods described herein. Sequential exposure ofbone marrow-derived hMSCs to hepatogenic factors results indifferentiation of the stem cells to cells with hepatocyte properties(see Snykers et al. BMC Dev Biol. 7:24, 2007; Aurich et al. Gut.56(3):405-15, 2007). Hepatogenic differentiation of bone marrow-derivedmesenchymal stem cells and adipose tissue-derived stem cells (ADSCs) hasalso been described (see Talens-Visconti et al. World J Gastroenterol.12(36):5834-45, 2006). Human hepatocytes can also be generated frommonocytes. Ruhnke et al. (Transplantation 79(9):1097-103, 2005) describethe generation of hepatocyte-like (NeoHep) cells from terminallydifferentiated peripheral blood monocytes. The NeoHep cells resembleprimary human hepatocytes with respect to morphology, expression ofhepatocyte markers, various secretory and metabolic functions and drugdetoxification activities. In addition, human hepatocytes derived fromamniocytes, also can be used in the methods described herein.

Hepatocytes can also be derived by reprogramming of distinct cell typessuch as skin fibroblasts (Huang et al., Nature 475(73560:386-389, 2011).

Human ES cell lines exist and can be used in the methods disclosedherein. Human ES cells can also be derived from preimplantation embryosfrom in vitro fertilized (IVF) embryos. Experiments on unused humanIVF-produced embryos are allowed in many countries, such as Singaporeand the United Kingdom, if the embryos are less than 14 days old. Onlyhigh quality embryos are suitable for ES cell isolation. Cultureconditions for culturing the one cell human embryo to the expandedblastocyst have been described (see, for example, Bongso et al., HumReprod. 4:706-713, 1989). Co-culturing of human embryos with humanoviductal cells results in the production of high blastocyst quality.IVF-derived expanded human blastocysts grown in cellular co-culture, orin improved defined medium, allows isolation of human ES cells (see U.S.Pat. No. 6,200,806).

C. Collecting Heterologous Hepatocytes from Engrafted Rats

Expanded hepatocytes can be collected from recipient rats using any of anumber of techniques known in the art. For example, rats can beanesthetized and the portal vein or inferior vena cava cannulated with acatheter. The liver can then be perfused with an appropriate buffer(such as a calcium- and magnesium-free EBSS supplemented with 0.5 mMEGTA and 10 mM HEPES), followed by collagenase treatment (for example,using a solution of EBSS with calcium and magnesium supplemented with 1mg/ml collagenase II). The digested liver is removed from the animals,minced to dissociate cellular aggregates and generate a homogenousslurry. To enrich for hepatocytes the cell slurry is passed throughnylon mesh (such as sequentially through 100 μm, 70 μm and 40 μm nylonmesh), followed by low speed centrifugation and washing of the cells.

Heterologous hepatocytes collected from recipient rats can be separatedfrom rat cells or other contaminants (such as tissue or cellular debris)using any technique well known in the art. For example, such methodsinclude using an antibody that selectively binds to the heterologoushepatocyte species. For human hepatocytes, such antibodies include, butare not limited to an antibody that specifically binds to a class Imajor histocompatibility antigen, such as anti-human HLA-A,B,C (Markuset al. Cell Transplantation 6:455-462, 1997) or CD46. Antibodiesspecific for human cells or human hepatocytes can be used in a varietyof different techniques, including fluorescence activated cell sorting(FACS), panning or magnetic bead separation. Alternatively, antibodieswhich bind selectively to the rat cells can be used to removecontaminating animal cells and thereby enrich human hepatocytes. FACSemploys a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cells (seeU.S. Pat. No. 5,061,620) bound by the antibody. Magnetic separationinvolves the use of paramagnetic particles which are: 1) conjugated tothe human-specific antibodies; 2) conjugated to detection antibodieswhich are able to bind to the human-specific antibodies; or 3)conjugated to avidin which can bind to biotinylated antibodies. Panninginvolves a monoclonal antibody attached to a solid matrix, such asagarose beads, polystyrene beads, hollow fiber membranes or plasticpetri dishes. Cells that are bound by the antibody can be isolated froma sample by simply physically separating the solid support from thesample.

Hepatocytes collected from rats can be, for example, cryopreserved forlater use, or plated in tissue culture for shipping and future use.

D. Liver Reconstitution

The rats described herein provide a system for propagating hepatocytesthat can be used to reconstitute a liver, as an alternative or adjunctto a liver transplant in a human or non-human mammal. Currently,patients suffering from liver disease may have to wait for long periodsof time before a suitable organ for transplant becomes available. Aftertransplant, patients need to be treated with immunosuppressive agentsfor the duration of their lives in order to avoid rejection of thedonor's liver. A method for propagating the patient's own remaininghealthy liver cells could provide a source of functional liver tissuewhich would not require immunosuppression to remain viable. Accordingly,the rats disclosed herein can be used to reconstitute the liver cells ofboth humans and non-human mammals with liver disease and/or liverfailure using their own hepatocytes, including those produced frompatient specific stem cells, that have been expanded in the rats, orhepatocytes from a donor.

Reconstitution of liver tissue in a patient by the introduction ofhepatocytes (also referred to as “hepatocyte transplantation”) is apotential therapeutic option for patients with acute liver failure,either as a temporary treatment in anticipation of liver transplant oras a definitive treatment for patients with isolated metabolicdeficiencies (Bumgardner et al., Transplantation 65: 53-61, 1998). Amajor obstacle to achieving therapeutic liver reconstitution is immunerejection of transplanted hepatocytes by the host, a phenomenon referredto (where the host and donor cells are genetically and phenotypicallydifferent) as “allograft rejection.” Immunosuppressive agents have beenonly partially successful in preventing allograft rejection (Javregui etal., Cell Transplantation 5: 353-367, 1996; Makowka et al.,Transplantation 42: 537-541, 1986).

Heterologous hepatocytes expanded in rats may also be used for genetherapy applications. In the broadest sense, such hepatocytes aretransplanted into a host to correct a genetic defect. The passagedhepatocytes need not, but can be derived originally from the sameindividual or subject who is to be the recipient of the transplant.

In some embodiments, heterologous hepatocytes expanded in rats may beused to reconstitute liver tissue in a subject as a prelude or analternative to liver transplant. As one non-limiting example, a subjectsuffering from progressive degeneration of the liver, for example, as aresult of alcoholism, may serve as a donor of hepatocytes that are thenexpanded in a rat. The number of hepatocytes is expanded relative to thenumber originally obtained from the subject and transplanted into therat. Following expansion, the expanded hepatocytes can be isolated fromthe rat, and can be used to reconstitute the subject's liver function.Expanding hepatocytes in rats may be used not only to increase thenumber of hepatocytes, but also to selectively remove hepatocytes thatare afflicted with infectious or malignant disease. Specifically, asubject may be suffering from hepatitis, where some but not all of thehepatocytes are infected and infected hepatocytes can be identified bythe presence of viral antigens in or on the cell surface. In such aninstance, hepatocytes can be collected from the subject, andnon-infected cells can be selected for expanding in one or more rats,for example by FACS. Meanwhile, aggressive steps could be taken toeliminate infection in the patient. Following treatment, the subject'sliver tissue may be reconstituted by hepatocytes expanded in the one ormore rats. An analogous method could be used to selectively passagenon-malignant cells from a patient suffering from a liver malignancy,such as HCC.

E. Models of HT1

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessivemetabolic disease that affects the liver and kidneys. HT1 results fromdefects in the Fah gene, located in q23-q25 of chromosome 15 in humansand in chromosome 7 in mice. HT1 patients display a variety of clinicalsymptoms, such as liver damage from infancy that advances to cirrhosis;reduced coagulation factors; hypoglycemia; high concentrations ofmethionine, phenylalanine, and aminolevulinic acid in serum plasma; highrisk of hepatocellular carcinoma; and tubular and glomerular renaldysfunction. In its severe form, a pattern of progressive liver damagebegins from early infancy. In its mild form, chronic liver damage with ahigh incidence of hepatoma is characteristic.

Animals homozygous for Fah gene disruption have a neonatal lethalphenotype caused by liver dysfunction. It has previously beendemonstrated that treatment of Fah-deficient mice with NTBC restoresliver function and abolishes neonatal lethality (Grompe et al., NatGenet 10:453-460, 1995). The prolonged lifespan of these animalsresulted in a phenotype analogous to HT1 in humans, including thedevelopment of hepatocellular carcinoma and fibrosis. Accordingly, theFah-deficient rats disclosed herein also represent animal models of thehuman disease HT1.

F. Liver Disease Model

As discussed above, Fah deficiency in rats leads to a disease phenotypesimilar to the human disease HT1. To prevent lethality, Fah-deficientrats are maintained on NTBC or another compound that avoids liverdysfunction, however, titration of the dose of NTBC can be used promotethe development of HT1-type phenotypes, including HCC, fibrosis andcirrhosis. Accordingly, the rats with various hepatic deficienciesdisclosed herein can be used to study a variety of liver diseases,including HCC and cirrhosis.

In some embodiments, the rats with humanized livers are used as animalmodels of human liver disease. The rats may be used as models of liverdisease resulting from, for example, exposure to a toxin, infectiousdisease or malignancy or a genetic defect (i.e., Fah-deficiency leadingto HT1). Examples of human genetic liver diseases for which the rats mayserve as a model include, but are not limited to, hypercholesterolemia,hypertriglyceridemia, hyperoxaluria, phenylketonuria, maple syrup urinedisease, glycogen storages diseases, lysosomal storage diseases (such asGaucher's disease), and any inborn error of metabolism. The disclosedmodel systems can be used to gain a better understanding of particularliver diseases and to identify agents which may prevent, retard orreverse the disease processes.

Where the rat is to be used as a model for liver disease caused by atoxin, the Fah-deficient rat is maintained on a dosage of NTBC (or adrug having a liver protective effect similar to NTBC) effective toinhibit liver dysfunction or liver failure. The amount of toxic agentrequired to produce results most closely mimicking the correspondinghuman condition may be determined by using a number of rats exposed toincremental doses of the toxic agent. Examples of toxic agents include,but are not limited to, ethanol, acetaminophen, phenytoin, methyldopa,isoniazid, carbon tetrachloride, yellow phosphorous and phalloidin. Insome cases, the rats, in the absence of human hepatocytes, are used asthe model for evaluating the effect of a toxin. In other examples, therat is transplanted with human hepatocytes to evaluate the effect of thetoxin on human hepatocytes. In these examples, it is not necessary tomaintain the Fah-deficient rat on a dosage of a liver protective drugsuch as NTBC to preserve liver function. Typically, expansion of humanhepatocytes is allowed to proceed to the point where the size of thehuman hepatocyte population is substantial (e.g. has approached amaximum), before the rat is exposed to the toxic agent.

In embodiments where a rat is to be used as a model for malignant liverdisease (such as HCC or hepatoma), the Fah-deficient rat is administereda high enough dose of a liver protective drug such as NTBC to preventfatality due to liver dysfunction, but low enough to allow thedevelopment of HCC or other liver malignancy. Alternatively, theFah-deficient rat can be maintained on a dose of the drug that preservesliver function and the malignancy can be produced by exposure to atransforming agent or by the introduction of malignant cells. In someexamples, the rat, in the absence of heterologous hepatocytes, is usedas the model for malignant liver disease. In other examples, the rat istransplanted with human hepatocytes to evaluate malignant liver diseaseof the human cells. In these examples, it is not necessary to maintainthe Fah-deficient rats on the liver protective drug, such as NTBC. Thetransforming agent or malignant cells may be introduced with the initialcolonizing introduction of human hepatocytes or after the humanhepatocytes have begun to proliferate in the host rat. In the case of atransforming agent, it may be preferable to administer the agent at atime when heterologous hepatocytes are actively proliferating. Examplesof transforming agents include aflatoxin, dimethylnitrosamine, and acholine-deficient diet containing 0.05-0.1% w/w DL-ethionine (Farber andSarma, 1987, in Concepts and Theories in Carcinogenesis, Maskens et al.,eds, Elsevier, Amsterdam, pp. 185-220). Such transforming agents may beadministered either systemically to the rat or locally into the liveritself. Malignant cells may be inoculated directly into the liver.

G. Model for Hepatic Infections

Hepatocytes expanded in and collected from recipient rats can also beused for a variety of microbiological studies. A number of pathogens(e.g., bacteria, viruses and parasites) will only replicate in a humanhost or in primary human hepatocytes. Thus, having a sufficient sourceof primary human hepatocytes is critical for studies of these pathogens.The expanded human hepatocytes can be used for studies of viralinfection and replication or for studies to identify compounds thatmodulate infection of hepatic viruses. Methods for using primary humanhepatocytes in studies of hepatic viruses are described in, for example,European Patent No. 1552740, U.S. Pat. No. 6,509,514 and PCT PublicationNo. WO 00/17338. Examples of hepatic viruses include hepatitis A virus,hepatitis B virus (HBV), hepatitis C virus (HCV) and cytomegalovirus(CMV). Examples of parasites that infect the liver include, for example,the causative agents of malaria (Plasmodium species, includingPlasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae and Plasmodium knowlesi) and the causative agents ofleishmaniasis (Leishmania species, including L. donovani, L. infantum,L. chagasi, L. mexicana, L. amazonensis, L. venezuelensis, L. tropica;L. major; L. aethiopica, L. (V.) braziliensis, L. (V.) guyanensis, L.(V.) panamensis, and L. (V.) peruviana).

In addition to using the human hepatocytes expanded in rats formicrobiological studies, the rats themselves can serve as animal modelsof hepatic pathogen infection. For example, rats repopulated with humanhepatocytes can be infected with a hepatic pathogen and used to screencandidate agents for treatment of the infection. Candidate agentsinclude any compound from any one of a number of chemical classes, suchas small organic compounds. Candidate agents also include biomolecules,such as, for example, nucleic acid molecules (including antisenseoligonucleotides, small interfering RNAs, microRNAs, ribozymes, shorthairpin RNAs, expression vectors and the like), peptides and antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate agents can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Using rats to study HCV and HBV infection, as well as evaluate candidateagents for the treatment of these infections, is discussed below.However, the methods can be applied to any hepatic pathogen of interest.In one embodiment, a rat is used to identify agents that inhibit viralinfection, decrease viral replication, and/or ameliorate one or moresymptoms caused by HBV or HCV infection. In general, the candidate agentis administered to the rat, and the effects of the candidate agentassessed relative to a control. For example, the candidate agent can beadministered to an HCV-infected rat model, and the viral titer of thetreated rat (e.g., as measured by RT-PCR of serum samples) can becompared to the viral titer of the rat prior to treatment and/or to anuntreated HCV-infected rat. A detectable decrease in viral titer of aninfected rat following treatment with a candidate agent is indicative ofantiviral activity of the agent.

The candidate agent can be administered in any suitable mannerappropriate for delivery of the agent. For example, the candidate agentcan be administered by injection (such as by injection intravenously,intramuscularly, subcutaneously, or directly into the target tissue),orally, or by any other desirable means. In some cases, the in vivoscreen will involve a number of rats receiving varying amounts andconcentrations of the candidate agent (from no agent to an amount ofagent that approaches an upper limit of the amount that can be safelydelivered to the animal), and may include delivery of the agent indifferent formulations and routes. Candidate agents can be administeredsingly or in combinations of two or more, especially whereadministration of a combination of agents may result in a synergisticeffect.

The activity of the candidate agent can be assessed using any one of avariety of means known in the art. For example, where the rat isinfected with a hepatotropic pathogen (e.g., HBV or HCV), the effect ofthe agent can be assessed by examining serum samples for the presence ofthe pathogen (e.g., measuring viral titer) or markers associated withthe presence of the pathogen (e.g., a pathogen-specific protein orencoding nucleic acid). Qualitative and quantitative methods fordetecting and assessing the presence and severity of viral infection arewell known in the art. In one embodiment, the activity of an agentagainst HBV infection can be assessed by examining serum samples and/ortissue sections for the presence of a viral antigen (such as HBV surfaceantigen (HBsAg) or HBV core antigen (HbcAg)). In another embodiment, theactivity of an agent against viral infection can be assessed byexamining serum samples for the presence of viral nucleic acid (such asHCV RNA). For example, HCV RNA can be detected using, for example,reverse transcriptase polymerase chain reaction (RT-PCR), competitiveRT-PCR or branched-DNA (bDNA) assay, detection of negative-strand RNA(the replicative intermediate of HCV) by RT-PCR, or sequencing of viralRNA to detect mutation/shift in the viral genome (“quasispeciesevolution”) with therapy. Alternatively or in addition, the host livermay be biopsied and in situ RT-PCR hybridization performed todemonstrate directly any qualitative or quantitative alterations in theamount of viral particles within tissue sections. Alternatively or inaddition, the host can be euthanized and the liver examinedhistologically for signs of infection and/or toxicity caused by theagent.

The rat models described herein can also be used to screen candidatevaccines for their ability to prevent or ameliorate infection by ahepatotropic pathogen. In general, a vaccine is an agent that, followingadministration, facilitates the host in mounting an immune responseagainst the target pathogen. The humoral, cellular, or humoral/cellularimmune response elicited can facilitate inhibition of infection by thepathogen against which the vaccine is developed. Of particular interestin the present disclosure are vaccines that elicit an immune responsethat inhibits infection by and/or intrahepatic replication of ahepatotropic pathogen (e.g., a microbial, viral, or parasitic pathogen),particularly a viral pathogen, such as HBV and/or HCV.

To evaluate candidate vaccines, the rats are transplanted with humanhepatocytes to repopulate the rat liver with human hepatocytes.Screening for an effective vaccine is similar to the screening methodsdescribed above. In some embodiments, the candidate vaccine isadministered to the rat prior to inoculation with the hepatotropicpathogen. In some cases, the candidate vaccine is administered byproviding a single bolus (e.g., intraperitoneal or intramuscularinjection, topical administration, or oral administration), which isoptionally followed by one or more booster immunizations. The inductionof an immune response can be assessed by examining B and T cellresponses that are specific for the antigen/vaccine according to methodswell known in the art. The immunized rat is then challenged with thehepatotropic pathogen. Typically, several immunized rats are challengedwith increasing titers of the pathogen. The rats are then observed fordevelopment of infection, and the severity of infection is assessed(such as by assessing the titer of the pathogen present, or examininghuman hepatocyte function parameters). Vaccine candidates that providefor a significant decrease in infection by the pathogen and/or asignificant decrease in the severity of disease that resultspost-challenge are identified as viable vaccines.

H. Pharmacology, Toxicology and Gene Therapy Studies

The described rat models and/or heterologous hepatocytes expanded in andcollected from the rats can be used to evaluate alterations in geneexpression in such hepatocytes by any pharmacologic compound, such assmall molecules, biologicals, environmental or biological toxins or genedelivery systems.

For example, human hepatocytes expanded in and collected from rats canbe used to evaluate toxicity of particular compounds in human cells.Methods of testing toxicity of compounds in isolated hepatocytes arewell known in the art and are described, for example, in PCT PublicationNo. WO 2007/022419. Similarly, rats transplanted with human hepatocytescan be used to evaluate the toxicity of exogenous agents. In someembodiments, the exogenous agent is a known or suspected toxin.

In some embodiments, rats transplanted with human hepatocytes (or humanhepatocytes expanded in and collected from rats) are used to evaluateany one of a number of parameters of drug metabolism andpharmacokinetics. For example, studies can be carried out to evaluatedrug metabolism, drug/drug interactions in vivo, drug half-life, routesof excretion/elimination, metabolites in the urine, feces, bile, bloodor other bodily fluid, cytochrome p450 induction, enterohepaticrecirculation, and enzyme/transporter induction.

In some embodiments, rats transplanted with heterologous hepatocytes (orheterologous hepatocytes expanded in and collected from rats) are usedto evaluate toxicology and safety of a compound, including therapeuticagents or candidate agents (such as small molecules or biologicals),environmental or biological toxins, or gene delivery systems. Forexample, cell cycle proliferation in expanded human hepatocytes can beevaluated, such as to determine the risk of cancer following exposure tothe compound. Toxicity to hepatocytes can also be assessed, such as byhistology, apoptosis index, liver function tests, gene expressionanalysis, metabolism analysis and the like. Analysis of hepatocytemetabolism can also be performed, such as analysis of metabolites afterinfection of stable isotope precursors.

The efficacy of particular drugs can also be evaluated in ratstransplanted with human hepatocytes. Such drugs include, for example,drugs to treat hyperlipidemia/atherosclerosis, hepatitis and malaria.

In some embodiments, rats transplanted with human hepatocytes (or humanhepatocytes expanded in and collected from rats) are used to study genetherapy protocols and vectors. For example, the following parameters canbe evaluated: transduction efficiency of gene delivery vehiclesincluding viral and non-viral vectors; integration frequency andlocation of genetic payloads (integration site analysis); functionalityof genetic payloads (gene expression levels, gene knockdown efficiency);and side effects of genetic payloads (analysis of gene expression orproteomics in human hepatocytes in vivo).

IV. Vectors Encoding Urokinase

In some embodiments of the methods described herein, the rats areadministered a vector encoding urokinase prior to transplantation ofheterologous hepatocytes. Ectopic expression of urokinase induceshepatocellular death and subsequently promotes liver regeneration, andcan thus aid in the efficiency of hepatocyte engraftment (Lieber et al.,Proc Natl Acad Sci USA 92:6210-6214, 1995).

In one embodiment, the urokinase (also known as urokinase plasminogenactivator (uPA)) is the secreted form of human urokinase. In anotherembodiment, the urokinase is a modified, non-secreted form of urokinase(see U.S. Pat. No. 5,980,886). Any type of suitable vector forexpression of urokinase in animals is contemplated. Such vectors includeplasmid vectors or viral vectors. Suitable vectors include, but are notlimited to, DNA vectors, adenovirus vectors, retroviral vectors,pseudotyped retroviral vectors, AAV vectors, gibbon ape leukemia vector,VSV-G, VL30 vectors, liposome mediated vectors, and the like. In oneembodiment, the viral vector is an adenovirus vector. The adenovirusvector can be derived from any suitable adenovirus, including anyadenovirus serotype (such as, but not limited to Ad2 and Ad5).Adenovirus vectors can be first, second, third and/or fourth generationadenoviral vectors or gutless adenoviral vectors. The non-viral vectorscan be constituted by plasmids, phospholipids or liposomes (cationic andanionic) of different structures. In another embodiment, the viralvector is an AAV vector. The AAV vector can be any suitable AAV vectorknown in the art.

Viral and non-viral vectors encoding urokinase are well known in theart. For example, an adenovirus vector encoding human urokinase isdescribed in U.S. Pat. No. 5,980,886 and by Lieber et al. (Proc. Natl.Acad. Sci. U.S.A. 92(13):6210-4, 1995). U.S. Patent ApplicationPublication No. 2005-176129 and U.S. Pat. No. 5,585,362 describerecombinant adenovirus vectors and U.S. Pat. No. 6,025,195 discloses anadenovirus vector for liver-specific expression. U.S. Patent ApplicationPublication No. 2003-0166284 describes adeno-associated virus (AAV)vectors for liver-specific expression of a gene of interest, includingurokinase. U.S. Pat. Nos. 6,521,225 and 5,589,377 describe recombinantAAV vectors. PCT Publication No. WO 0244393 describes viral andnon-viral vectors comprising the human urokinase plasminogen activatorgene. An expression vector capable of high level of expression of thehuman urokinase gene is disclosed in PCT Publication No. WO 03087393.Each of the aforementioned patents and publications are hereinincorporated by reference.

Vectors encoding urokinase can optionally include expression controlsequences, including appropriate promoters, enhancers, transcriptionterminators, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns and maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons. Generally expression control sequences include a promoter, aminimal sequence sufficient to direct transcription.

The expression vector can contain an origin of replication, a promoter,as well as specific genes which allow phenotypic selection of thetransformed cells (such as an antibiotic resistance cassette).Generally, the expression vector will include a promoter. The promotercan be inducible or constitutive. The promoter can be tissue specific.Suitable promoters include the thymidine kinase promoter (TK),metallothionein I, polyhedron, neuron specific enolase, thyrosinehyroxylase, beta-actin, or other promoters. In one embodiment, thepromoter is a heterologous promoter.

In one example, the sequence encoding urokinase is located downstream ofthe desired promoter. Optionally, an enhancer element is also included,and can generally be located anywhere on the vector and still have anenhancing effect. However, the amount of increased activity willgenerally diminish with distance.

The vector encoding urokinase can be administered by a variety ofroutes, including, but not limited to, intravenously, intraperitoneallyor by intravascular infusion via portal vein. The amount of vectoradministered varies and can be determined using routine experimentation.In one embodiment, rats are administered an adenovirus vector encodingurokinase at a dose of about 1×10⁸ to about 1×10¹⁰ plaque forming units,such as about 5×10⁹ plaque forming units.

In one embodiment, the rat is administered an adenovirus vector encodinghuman urokinase. Adenovirus vectors have several advantages over othertypes of viral vectors, such as they can be generated to very hightiters of infectious particles; they infect a great variety of cells;they efficiently transfer genes to cells that are not dividing; and theyare seldom integrated in the guest genome, which avoids the risk ofcellular transformation by insertional mutagenesis (Douglas and Curiel,Science and Medicine, March/April 1997, pages 44-53; Zern and Kresinam,Hepatology:25(2), 484-491, 1997). Representative adenoviral vectorswhich can be used to, encode urokinase are described byStratford-Perricaudet et al. (J. Clin. Invest. 90: 626-630, 1992);Graham and Prevec (In Methods in Molecular Biology: Gene Transfer andExpression Protocols 7: 109-128, 1991); and Barr et al. (Gene Therapy,2:151-155, 1995).

Additional advantages of the described embodiments will be apparent tothose skilled in the art based upon the examples.

EXAMPLES

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Example 1 Generation of the AAV-rFAH:PGK-NEO Null Targeting Vector andProduction of FAH KO Rat ES Cells

The rat FAH KO construct targets exon 3 of the FAH gene. Rat ES cellgenomic DNA was used to generate 5′ and 3′ FAH homology regions for thetargeting construct. A 5′ FAH homology region was generated using aforward primer 1.5 kb upstream of exon 3 (intron 1-2; Table 1), and thereverse primer containing a stop codon ending 54 bp into exon 3 of theFAH gene (SEQ ID NO:1; FIG. 1). A 3′ FAH homology region was generatedusing a forward primer starting 90 bp into exon3 and ending in intron4-5 (1.5 kb; Table 1). The two 1.5 kb PCR products were cloned,sequenced and ligated to a PGK:NEO selection cassette totaling 4.7 kb(FIG. 2). The nucleotide sequence of the targeting cassette is set forthherein as SEQ ID NO:2.

The 4.7 kb fragment was ligated into the pcDNA 3.1 vector, which waslinearized and transfected into rat 208D fibroblast cells to examine NEOresistance. The cells were selected with 500 μg/mL G418 for 1 week.Resistant 208D clones were obtained. The 4.7 kb cassette was thenligated into the pAAV2 shuttle vector, and clones containing the entireFAH KO cassette were selected and the recombinant AAV was generated. TheAAV-rFAH:PGK-NEO construct was checked for intact internal repeatsequences and sequenced before being used for AAV production.

TABLE 1 Primers used in the design of rFAH construct SEQ Primer IDLocation Primer Sequence 5′ to 3′ NO: rFAH Intron  gtagcgaattcgcggccgc 3 1-2 FOR GCTGTGAGGTCAGAGACCAGCC rFAH Exon  ggatagaattc  4 3 REVGTCACCATGCCGCTTGGCCGAGGCCC rFAH Exon  gtagcaagctt   5 3 FORGCCAGCCAAGCCCAGCTCAGA rFAH Intron  ggataggtaccgcggccgc  6 3-4 REVGACCTCTAGTTCCATGTATGG 5′ FAH FOR  gaggccttgttcacacatga  7 genotyping 5′FAH REV  ctaaagcgcatgctccagac  8 genotyping 3′ FAH FOR attgcatcgcattgtctgag  9 genotyping 3′ FAH REV  agtctcctgcagagggaaca 10genotyping Neo geno- TGCTCCTGCCGAGAAAGTAT 11 typing FOR Neo geno-CAACAGATGGCTGGCAACTA 12 typing REV 5′ FAH  gtagcgaattcgcggccgc 13FOR (CAG) GCTGTGAGGTCAGAGACCAGCC 5′ FAH  tagtcgacgtcaaggatgctcttgcctcct14 REV (CAG) 3′ FAH  gcaggtggtgccacttgtccccagttgagg 15 FOR (CAG) 3′ FAH gtatgcatatcgatggaattcccctttcca 16 REV (CAG)

Purified, high titer virus was propagated and used to transduce ratembryonic stem cells. Recombinant FAH KO clones were selected using 100μg/mL of G418. The ES cell clones that were able to grow under theselection were further amplified and genomic DNA was isolated. ES cellclones from the 95 G418 selected clones integrated into the correctlocus of the rat genome as determined by PCR using FAH specific primersfor the 5′ end or 3′ end of the integration site. Ten ES clones werefurther validated by Southern blot analysis to confirm correct genomicintegration of the FAH KO cassette (FIG. 3). First the ES clones werethawed and expanded in duplicate wells of a 24 well plate. One well foreach clone was harvested, pelleted and frozen in liquid nitrogen, andthen stored at −80° C. until ready to use. Genomic DNA was isolated fromthe sample by lysing the cell pellet with SDS, digesting the protein andprecipitating the genomic DNA (gDNA) with saturated sodium chloride andisopropanol. Approximately 20 μg of gDNA from each clone was digestedwith the restriction endonuclease Bgl II, and the samples wereelectrophoresed on a 0.7% Agarose:Tris-borate-EDTA gel for 6 hours at 45volts. The DNA was exposed to acid depurination prior to capillarytransfer with alkaline transfer buffer. The recombined FRG KO cloneswere identified by hybridization of the nylon membrane using a ³²Plabeled probe that mapped immediately to the 3′ end of the FRGKO:PGK-Neomycin cassette. FIG. 4 is a schematic of the rFah gene withthe integrated targeting cassette. Knock-out recombinant clones wereidentified by a unique 7.9 Kb fragment. The results are shown in FIGS.5-6.

FIG. 5(a) confirms integration by homologous recombination. By using the5′ FAH+NEO (reverse) and 3′ FAH+NEO (forward) primers, it was confirmedthat both flanks were intact as predicted for integration into exon 3 byhomologous recombination. FIG. 5(b) is the control gel confirming thepresence of DNA in the samples. FIG. 6 shows a schematic of the rFahgene wild type and KO genome of the targeted region, along with theSouthern Blot using an Fah probe. Both the untargeted and targetedalleles are visualized and have the expected band sizes. This confirmsproper targeting of the rat Fah locus in clones 11, 15, and 18.

The targeted FAH knock-out ES cells can be used to generate an Fah KOrat using procedures reported by Tong et al. (Nature 467(7312):211-213,2010).

Example 2 Generation of FAH KO Rats and Transplantation of HumanHepatocytes

FAH KO rats were generated by injecting FAH KO ES cells into ratblastocysts, which are then transferred to E3.5 pseudo-pregnant SD rats.The chimeric progeny from these rats are bred to generate heterozygousFAH KO rats by germline transmission. Male and female heterozygotes arethen further bred to generate FAH knockout rats. The deletion of the FAHgene allows for the induction of liver damage to aid in high levels ofengraftment and repopulation of the liver with transplanted humanhepatocytes.

Prior to transplantation with human hepatocytes, acute liver damage isinduced in the rats by dosing with a recombinant adenovirus expressingthe plasminogen-like urokinase enzyme. Twenty-four hours later the humanhepatocytes are delivered via intra-splenic injection where thehepatocytes will travel through the vasculature to reach the liver. Inaddition, immune suppression drugs are given to the rats before, duringand after the transplant to eliminate the host versus graft response inthe rat from the xenografted human hepatocytes. By cycling the rats offNTBC for defined periods of time, the rat cells become quiescent and thehuman cells have a proliferative advantage, leading to replacement ofrat hepatocytes with human hepatocytes, and generating rats with highlevels of human chimerism in the liver. Human hepatocyte repopulationlevels are determined through the quantitation of human serum albuminlevels and other human specific markers and correlated withimmunohistochemistry of liver sections from transplanted rats.

Example 3 Generation of the Il2rg Construct

The rat Il2rg construct targets exon 3 of the Il2rg gene. Rat ES cellgenomic DNA was used to generate 5′ and 3′ Il2rg homology regions forthe targeting construct. A 5′ Il2rg homology region was generated usinga forward primer 1.46 kb upstream of exon 3 (Table 2), and a reverseprimer containing a stop codon ending 40 bp inside exon 3 of the IL2rggene. A 3′ Il2rg homology region was generated using a forward primercontaining 40 bp of exon3 and ending in exon 6 (1.5 kb; Table 2). Thetwo 1.5 kb PCR products were cloned, sequenced and ligated to a PGK:NEOselection cassette totaling 4.7 kb, similar to that of the FAH constructdescribed in Example 1. The construct was sequenced and used for viralproduction.

TABLE 2 Primers used in the design of the rIL2rg construct SEQ Primer IDLocation Primer Sequence 5′ to 3′ NO: 5′ Rat gtagcgaattcgcggccgctgattggatt 17 IL2rg FOR ctcggtgtga 5′ Rat ggatagaattcgtcagtggctgcactcct 18 IL2rg REV ggaatgtattatt 3′ Rat  gtagca 19 IL2rg FOR agcttaggcgagccgaacagaagctaaac 3′ Rat ggataggtaccgcggccgcCAGGGATAAG 20 IL2rg REV CACAGCTTCC

Example 4 Perfusion and Isolation of Hepatocytes from a Humanized Mouse

In this Example, human hepatocytes were isolated from a geneticallymodified mouse (the FRG KO mouse) repopulated with human hepatocytes.The human hepatocytes isolated from the mouse were used in the studiesdescribed in Example 5.

Hepatocytes were isolated from an in situ liver of a highly repopulatedmouse (FRG knockout mouse model, Yecuris Corporation; see WO 2008/151283and WO 2010/127275): albumin level=4.37 mg/mL, NTBC status: 0 mg/L)using a collagenase perfusion method. The mouse was first anaesthetizedand then immobilized on a moisture-absorbing surface. The abdomen wasopened and a cannula was inserted into the portal vein. The liver wasthen perfused with EBSS without calcium or magnesium, 10 mM Hepes pH 7.4and 0.5 mM EDTA to blanch the liver and eliminate any blood clotting.Next the liver was perfused using a 1 mg/mL collagenase Type II solutionin EBSS with calcium and magnesium and 10 mM Hepes pH 7.4 for 8 minutesuntil the liver was completely digested. The liver was then removed to apetri dish and dissociated using forceps and scissors to a homogenousslurry. Next, 5 mL of perfusion media (DMEM+10% fetal bovine serum) wasused to deactivate the collagenase.

Using a sterile 25 mL pipet, the liver slurry was passed through a100-micron filter into a 50 mL tube. The petri dish was then washed withperfusion media, passing it through the filter into the 50 mL tube. Thevolume in the tube was adjusted to 45 mL with perfusion media, andinverted two-three times to ensure a homogenous solution. The cellsuspension was then passed through a 70-micron filter into a fresh 50 mLtube. The cells were centrifuged for 5 minutes at 140×g and 4° C. Thesupernatant was carefully aspirated and the cell pellet was resuspendedin 10 mL of perfusion media. To dissociate any cell aggregates the cellsuspension was passed through the 10 mL pipet for a total of five times.The suspension volume was adjusted to 45 mL with perfusion media. Thecells were pelleted by centrifugation for 5 minutes at 140×g and 4° C.This is considered the first wash of the cell pellet. This step isrepeated one more time. After the last centrifugation the cells wereresuspended in 10 mL of perfusion media, passed through the pipet fivetimes, and the volume adjusted to 45 mL with perfusion media. The cellsuspension was then diluted 1:2 in perfusion media and counted withTrypan Blue in a hemocytometer chamber. The results are listed below:

Non- % # Viable Total viable Mouse Viable viable Total Viable ofcells/ml cells 1338 297 19 316 94 3 × 10⁶ 135 × 10⁶

Cells (2.25×10⁶) used for FACs analysis were transferred to a sterile 15mL tube and the volume was adjusted to 10 mL with perfusion media.

Mouse Volume (mL) 1338 0.75The following cell numbers were removed for transplant into rats:

# Cells/mouse Total # of mice Total mL Total # cells 1.00 × 10⁶ 15  3 mL15.00 × 10⁶ 3.00 × 10⁶ 17 17 mL 51.00 × 10⁶The volumes were equalized between the two tubes. The cells werepelleted at 140×g for 5 minutes at 4° C. The supernatant was aspiratedand the pellets were resuspended in the indicated volume of specifiedmedia:

Purpose Solution 1 Volume Total cell # FACs Perfusion media 850 μL  2.25× 10⁶ 1.00 × 10⁶ HCM (Lonza, 3.375 mL + 375 μL 15.00 × 10⁶ cells/mouseCat # C3198) Anakinra 3.00 × 10⁶ HCM (Lonza, 3.825 mL + 425 μL 51.00 ×10⁶ cells/mouse Cat # C3198) Anakinra

FACS Analysis to Determine % Human Hepatocytes:

4×200 μL of the cell suspension to be used for FACs analysis wasdispensed into 1.5 mL microfuge tubes (each tube contained ˜400,000cells). The following primary antibodies were then added to each tube:#1=nothing, negative control; #2=2 μL of HLA ABC; #3=2 μL of OC2-2F8 and2 μL of OC2-2G9; and #4=2 μL of HLA ABC, 2 μL of OC2-2F8 and 2 μL ofOC2-2G9.

The solution was mixed by flicking the tubes, followed by incubating onice for 30-60 minutes, with flicking every 5-10 minutes to keep cellssuspended. The primary antibody was washed out by adding 1 mL ofperfusion media to each tube and mixed by inversion several times. Thecells were pelleted by centrifugation at 100×g for 3 minutes at 4° C.The supernatant was aspirated, followed by resuspending each cell pelletin 200 μL of a secondary antibody solution consisting of: 850 μLperfusion media; 8.5 μL Strept-APC; and 8.5 μL anti-rat PE. The solutionwas mixed by flicking the tubes, followed by incubating on ice for 30-60minutes, with flicking every 5-10 minutes to keep cells suspended.

The secondary antibody was washed out by adding 1 mL of perfusion mediato each tube, followed by mixing by inverting. The tubes were thencentrifuged at 100×g for 3 minutes at 4° C., to pellet the cells,followed by aspirating the supernatant and resuspending each cell pelletin 200 μL. of PI solution: 5 μL PI; 4 μL 0.5M EDTA; and 1 mL perfusionmedia. FACs analysis determined the percentage of repopulation by humanhepatocytes in the mouse liver was 91%.

Example 5 Engraftment of Human Hepatocytes into Immunodeficient Rats

Liver humanization has not been successfully attempted in rats withoutthe use of partial hepatectomy. In this example, Nude (RNU) rates wererepopulated with human hepatocytes by injecting with Ad:uPa transplantedwith human hepatocytes that contained Anakinra and treated with FK506,without a partial hepatectomy.

Forty nude rat pups (2 weeks old) were obtained from Charles River andacclimated for a week before the study began. The rats were assignedto + or −immunosuppressive groups, with controls in each group (seeTable 3 below). Rats assigned to the +immunosuppressive group weretreated with 2 mg/kg/day subcutaneous doses of FK506 (Tacrolimus)throughout the study, with the initial dosage being 20 μl of 5 mg/mLFK506 (i.p. injection) per day, based upon an average initial animalweight of 50 g at the start of the study. The −immunosuppression ratswere not treated with FK506, but were injected subcutaneously with thesame volume of saline throughout the study. All of the rats were thengiven an intravenous injection (retroorbital) of uPA adenovirus (2.5×10⁹plaque forming units (PFU) per 50 g rat) 48-96 hours before hepatocyteinjection.

Twenty-one days after the first dose of FK506 was administered the ratswere then injected intrasplenically with placebo (PBS) or cellsuspensions of human hepatocytes with Anakinra, according to Table 3below, followed by post-operative administration of Buprenex stock at 1μg/mL (5 μL/g) and Cetiofur at 1.25 mg/mL (200 μL). The humanhepatocytes were perfused from humanized mouse livers as explained inExample 4.

TABLE 3 Animals and Cells Total Cell Rats + Rats − Conditions NumberImmunosuppression Immunosuppression Control 0 3 3 (0.2 mL PBS) 5 × 10⁶cells/mL 1 × 10⁶ 8 6 (0.2 mL) 10 × 10⁶ cells/mL 3 × 10⁶ 8 7 (0.3 mL)Total Rats 19 16 Total Cells 32 × 10⁶ 27 × 10⁶ Needed

Twenty-four hours after hepatocyte injection, all of the rats wereinjected with Anakinra (10 μL/2510 μL/25 g), Buprenex (5 μL/g) andCeftiofur at 1.25 mg/mL (200 μL), and then again at 48 hours post-op.Three days after surgery, 2 μL of blood was collected (saphenous-veinbleed) from each rat. The blood was diluted into 1 mL of ELISA buffersample diluent. Samples were then tested for human serum albumin (hSA)by Quantitative human Albumin ELISA. This process was repeated weeklythroughout the study. Analysis from the first ELISA showed that theaverage optical density reading (450 nm) of the experimental samples wastwice that of the PBS injected controls. Both the −FK506 injected with1×10⁶ cells and +FK-506 injected with 3×10⁶ cells had the highestoptical density readings at 0.027. Though the range of human albuminlevels fell below the linear range of the ELISA standard curve, thesamples still exhibited detectable optical density readings for humanalbumin.

Two weeks after study commencement, the dose of FK506 was increased to30 μl of 5 mg/mL due to an increase in animal weight. Four weeks afterstudy commencement, the dose of FK506 was again increased to 25 n1 of 10mg/mL to account for growth of the rats.

Four weeks after hepatocyte injection (˜5 weeks after studycommencement), at least 50 μl of whole blood was collected from the ratsand allowed to clot for 1 hour at room temperature. Serum was collectedby centrifuging the whole blood at 1,500 rpm×15 minutes. The serum wasthen diluted 1:25 and 1:50 and used for hSA testing via ELISA. Five μlof whole blood was also taken from 6 random rats in the +FK506 treatmentgroup. Five animals tested positive for human albumin between the rangesof 12 and 70 ng/mL with serum. One whole blood sample (#33) testedpositive for human albumin at 220 ng/mL. The rats were also reweighedand it was found that the +immunosuppressed rats were significantlysmaller than their non-treated counterparts, therefore theadministration of FK506 was changed to every other day.

Eight weeks after hepatocyte injection, 5 μl of whole blood was takenfrom each animal for ELISAs. The blood was immediately diluted in 1 mLof sample buffer for a dilution of 1:200. Rat #29 (3×10⁶; +FK506 group)tested positive for hSA in all replicates. Results were confirmed by anindependent blind repeat.

Almost nine weeks after hepatocyte injection, all animals weresacrificed. Cardiac punctures and exsanguinations were performed toobtain whole blood and serum for ELISAs. Animals were first dosed with500 μL of a ketamine cocktail before cardiac puncture. Portions of thespleen and liver (lobe 6) were flash frozen for genomic DNA isolationand detection of human genes. The remaining liver and spleen portionswere then fixed in 10% normal buffered formalin for 48 hours, at whichtime they were transferred to 70% ethanol prior to paraffin embedding.Liver sections were stained with FAH antibody to identify the humanhepatocytes. A humanized FRG KO mouse liver was used as a control.Tissues were analyzed for histologic evaluation and staining. Theresults are shown in FIG. 7. Sections were generated from a controlliver and spleen as well as the liver and spleen from rat #29. Theimages show that the liver from rat #29 contained positive humanhepatocytes by staining with human specific FAH antibody. These sectionsare similar to the engraftment observed in the confirmed FAH mouse modelwith the same antibody.

The initial ELISA data demonstrated the presence of human serum albuminin the engrafted nude rats. The data obtained from subsequent ELISAanalyses, as well as the positive IHC indicate that a population ofhuman cells were able to engraft into nude rats treated with uPA,Anakinra, and FK506. The levels of human albumin (˜1.5 μg/mL) relate to10,000 hepatocytes in the liver being human hepatocytes.

Example 6 Fah-Deficient Rats

Rats (Sprague Dawley strain) with mutations in the highly conserved Fahgene were generated using Transcriptional Activator-Like EffectorNucleases (TALENs) designed to target the endogenous Rattus norvegicusFah gene exon 3 sequence (SEQ ID NO:21; FIG. 8), where the underlinedportions of the sequence are the target binding sequences of two TALENmonomers, on opposite DNA strands, separated by the lowercase sequencewhere the TALEN heterodimer-mediated cleavage occurs. Standardmicroinjection of the male pronucleus of rat embryos from the Dahl SS(SS; SS/JrHsdMcwi) and Sprague Dawley (SD; SD/Crl) with an equimolarmixture of two in vitro-transcribed mRNAs encoding each TALEN monomer ata final concentration of 10 ng/μL led to the identification of threefounders with putative disruptive mutations in the Fah gene using theSurveyor Nuclease Mutation Detection kit (Transgenomic, Inc) aspreviously described (Geurts, A. M. et al. Generation of gene-specificmutated rats using zinc-finger nucleases. Methods Mol Biol 597, 211-25(2010)).

Disruption of the rat Fah target gene was confirmed using Sangersequencing and all three mutant animal alleles were evidenced by microdeletions (1-20 base pairs) of coding sequence within exon 3 (FIG.8(b)). Three mutant alleles were identified (Fah-m1, Fah-m2 and Fah-m3).Fah-m1 (6-bp deletion) and Fah-m2 (20-bp deletion) were generated in theSS while Fah-m3 (1-bp deletion) was identified in the SD rat straingenetic backgrounds. Two mutations (Fah-m2 and Fah-m3) cause aframeshift in the nascent gene transcript shifting the natural openreading frame after Serine 69 or Glycine 73, respectively, andtruncation of the protein product via the insertion of stop codons intothe reading frame after amino acids 91 and 120, respectively. The Fah-m1mutation results in a loss of three amino acids and insertion of oneamino acid (Methionine 71-Glycine 72-Lysine 73) and insertion of anisoleucine residue (FIG. 9; SEQ ID NOS:22-25) from these founders, threecolonies of mutant animals (SS-Fah-m1, SS-Fah-m2 and SD-Fah-m3) wereestablished by backcross and intercross of heterozygous carriers.

Mutations in the Il2rg and Rag2 genes were generated in similar fashionin both genetic backgrounds. Target sequences and alleles are shown inFIG. 10 (SEQ ID NOs:26-27). All mutations were predicted to beframe-shift truncations of the target gene. Knockout of both genes onthe SS genetic background leads to severe immune deficiency of T- andB-cell populations in both strains and NK-cell populations arediminished in the SS-Il2rg-m1 strain (FIGS. 11-12).

Disruption of Fah in the rat for all three alleles leads to embryoniclethality as no homozygous offspring are observed in intercrosses ofheterozygous animals in the absence of any treatment. Administering NTBCto the drinking water of breeding heterozygotes (8 mg/mL), however, canrescue the lethal effects of these mutations at Mendelian ornear-Mendelian ratios, depending on the allele (FIG. 13), demonstratingthat this drug can protect mutant rat embryos from the embryoniclethality. SS-Fah-m1 null animals maintained on NTBC in their drinkingwater are healthy and are reproductively fit. The Table in FIG. 13 showsthe genotypes of live born rat pups from heterozygous breedings. Thenumber of pups with each genotype expected from Mendellian ratios isshown, along with the actually observed numbers in red. Without NTBCtreatment during pregnancy, no homozygous mutant pups were born.Addition of NTBC to the drinking water of the mothers resulted in thebirth of mutant embryos with both Fah-m1 and Fah-m2 breeders.

Withdrawal of NTBC from an adult animal SS-Fah-m1 animal, but otherwisead libitum access to food and water, leads to a rapid decline in bodyweight (>20% loss in 7-8 days). Examination of fixed liver tissue bysections and trichrome staining (FIG. 14) revealed marked vacuolizationand enlargement of the hepatocytes. In addition, necrotic hepatocytesare seen in high numbers, whereas none were seen in the control,NTBC-treated littermate. Similarly, the kidneys showed extensive injury,especially cystic dilatation of the tubules, after NTBC withdrawal (FIG.15). These histological findings are typical for Fah deficiency inrodents.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A genetically modified Fah-deficient rat whose genome is homozygousfor a disruption in the Fah gene such that the disruption results inloss of expression of functional FAH protein and decreased liverfunction.
 2. The Fah-deficient rat of claim 1, wherein the rat isimmunosuppressed.
 3. The Fah-deficient rat of claim 2, wherein theimmunosuppression is the result of administration of one or moreimmunosuppressive agents.
 4. The Fah-deficient rat of claim 3, whereinthe one or more immunosuppressive agents are selected from the groupconsisting of FK506, cyclosporin A, fludarabine, mycophenolate,prednisone, rapamycin, azathioprine, and combinations thereof.
 5. TheFah-deficient rat of claim 2, wherein the immunosuppression is theresult of one or more genetic alterations that inhibit the developmentof functional immune cells.
 6. The Fah-deficient rat of claim 5, whereinthe one or more genetic alterations is selected from the groupconsisting of Rag1 deficiency, Rag2 deficiency, Il2rg deficiency, SCID,Sirp-α^(hum/hum), nude, perforin knockouts, and combinations thereof. 7.The Fah-deficient rat of claim 1, wherein the rat comprises transplantedheterologous hepatocytes.
 8. The Fah-deficient rat of claim 7, whereinthe heterologous hepatocytes are mammalian hepatocytes from a mammalselected from the group consisting of dogs, pigs, horses, rabbits, mice,marmoset, woodchuck, non-human primates, and humans.
 9. TheFah-deficient rat of claim 1, wherein the rat comprises expanded humanhepatocytes, wherein liver function is restored in the rat.
 10. A methodof expanding human hepatocytes in vivo, comprising: transplantingheterologous hepatocytes into a first rat, wherein said first rat is: agenetically modified Fah-deficient rat whose genome is homozygous for adisruption in the Fah gene such that the disruption results in loss ofexpression of functional FAR protein and decreased liver function, or animmunodeficient rat, wherein the immunodeficiency of the rat is due to agenetic alteration, immunosuppression, or a combination thereof, andallowing the heterologous hepatocytes to expand, thereby expandingheterologous hepatocytes in vivo.
 11. The method of claim 10, whereinthe heterologous hepatocytes transplanted into the first rat areisolated human hepatocytes.
 12. The method of claim 10, wherein thehuman hepatocytes are isolated from the liver of a humanized non-humanmammal prior to transplanting into the first rat, wherein the non-humanmammal is an FRG KO mouse.
 13. The method of claim 10, furthercomprising inducing acute liver damage in the first rat prior totransplanting the heterologous hepatocytes.
 14. The method of claim 10,further comprising administering a vector encoding urokinase to thefirst rat prior to transplanting the heterologous hepatocytes.
 15. Themethod of claim 10, wherein the first rat is an Fah-deficient rat,further comprising administering one or more immunosuppressive agents tothe first Fah-deficient rat at least about 2 days prior to transplantingthe heterologous hepatocytes.
 16. The method of claim 10, wherein thefirst rat is an immunodeficient rat, wherein the immunodeficiency is dueto one or more genetic alterations that inhibit the development offunctional immune cells, wherein the one or more genetic alterations isselected from the group consisting of selected from the group consistingof Rag1 deficiency, Rag2 deficiency, Il2rg deficiency, SCID,Sirp-α^(hum/hum), nude, perforin^(−/−), and combinations thereof. 17.The method of claim 10, further comprising collecting the expandedheterologous hepatocytes from the first rat.
 18. The method of claim 17,further comprising expanding the collected heterologous hepatocytes byserial transplantation, wherein said serial transplantation comprises:collecting expanded heterologous hepatocytes from the first rat;transplanting the collected expanded heterologous hepatocytes from thefirst rat into a second rat wherein said second rat is: a geneticallymodified Fah-deficient rat whose genome is homozygous for a disruptionin the Fah gene such that the disruption results in loss of expressionof functional FAH protein and decreased liver function, or animmunodeficient rat, wherein the immunodeficiency of the rat is due to agenetic alteration, immunosuppression, or a combination thereof; andallowing the heterologous hepatocytes to expand, thereby expandingheterologous hepatocytes in vivo.
 19. An immunodeficient rat having aliver comprising transplanted and expanded human hepatocytes, whereinthe immunodeficiency of the rat is due to a genetic alteration,immunosuppression, or a combination thereof.
 20. The immunodeficient ratof claim 19, wherein the immunodeficiency is due to one or more geneticalterations selected from the group consisting of selected from thegroup consisting of Rag1 deficiency, Rag2 deficiency, Il2rg deficiency,SCID, Sirp-α^(hum/hum), nude, perforin^(−/−), and combinations thereof.